Microfabrication of membranes for the growth of cells

ABSTRACT

The present invention provides a novel substrate for use in growing cells and for the study of mechanobiology. The membrane of the present invention comprises appropriate microtopography and surface chemical modifications to facilitate the production of adherent and oriented cells that phenotypically resemble cells in vivo.

[0001] The present application claims the benefit of priority of U.S.Provisional application No. 60/235,094 filed Sep. 25, 2000. The entiretext of that specification is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of cellgrowth and culture. More particularly, the present invention providesnovel methods and compositions for the growth of cells in ananatomically correct adult phenotype in vitro.

BACKGROUND

[0003] Cells in the body respond to extracellular stimuli, that are bothbiochemical and mechanical in nature (Vandenburgh, Am. J. Physiol262:R350-355, 1992; Buckley, Bone Miner. 4:225-236, 1988; Brunette, CellScience, 69:35-45, 1984; Harris, 3. Biomech Engineering, 106:19-24,1984). Both endothelium and muscle respond dynamically to mechanicalstimuli and serve as signal transduction interfaces. Although a muchfocused research topic in cell physiology, there are some fundamentalissues in experimental set-up of muscle cell cultures which have notbeen adequately addressed.

[0004] Mechanobiological studies usually involve statically strainedmembranes upon which cell monolayers are grown. However, such in vitroapproaches are ineffective at providing a good indication of cellfunction in vivo for a number of reasons. Firstly, these cell culturesystems produce significant detachment between the membrane that isbeing stretched and the overlying substrata. Secondly, unlike thecomplex three-dimensional force effects seen in vivo, the traditional invitro culture systems forces are transmitted in only one direction.Furthermore, the complex three-dimensional arrangement of myocytes, andin particular, cardiac myocytes as found in vivo, is usually lacking inthe in vitro models. Therefore, in understanding the role of mechanicalstimuli upon cell functional processes in culture, it would bebeneficial to provide an appropriate membrane or matrix that will moreclosely mimic the in vivo cellular arrangement.

[0005] An example of this can be seen in studies examining the effectsof stretch on cardiac gene regulation. In such experiments, myocytes,usually rat cardiac myocytes, are grown in monolayer culture uponsilicone and subjected to external mechanical stress. There have beenstudies of cardiac myocytes, in which the rate of protein synthesis fornon-aligned cells has been measured using silicone membranes that usedcollagen to keep cells attached. (Terracio et al., In Vitro Cellular andDevelopmental Biology, Vol. 24, 1988; Sharp et al., Circ. Res. 73:172-183,1993; Am. J. Physiol, 42: H546-H556, 1997). However, even thoughmyocytes do adhere to collagen quite well in static culture, there arestill significant problems with detachment of the collagen layer fromthe silicone substrate upon repeated mechanical deformation. It is notsurprising that this occurs, especially since it is well establishedthat proteins and cells do not exhibit good adherence to smooth, lowsurface energy materials such as silicone.

[0006] To date, primary neonatal cultures have been the mainstay in thestudy of myocyte function since contractile cardiac cell lines are notavailable. However, when it comes to the study of the contractilefunction and processes of assembly primary neonatal cells are woefullyinadequate since they generally have very few functioning myofibrils.Contractile activity is clearly an important signal in regulation ofmyocyte cell shape that leads, in turn, to remodeling the shape andfunction of the whole heart. Unfortunately, most adult and neonatalmyocyte culture systems display little or no contractile activity.

[0007] Thus, there is a need for phenotypically normal myocytes that canbe manipulated experimentally. Furthermore there is a need to develop aculture substrata that allows cells to adhere and remain adhered duringthe application of mechanical and other force.

SUMMARY OF THE INVENTION

[0008] The present invention provides a novel substrata for use ingrowing cells. The membrane of the present invention comprisesappropriate microtopography and surface modifications to facilitate theproduction of adherent and oriented cells that phenotypically resemblecells in vivo. Particularly preferred for the present invention aremuscle cells.

[0009] Specifically, the present invention contemplates a biocompatible,deformable membrane for the growth of cells e.g., muscle cells,comprising a microtextured polymer membrane having projections ofbetween about 1 μm to about 100 μm in size and longitudinal grooves;wherein the polymer membrane comprises a surface modification tofacilitate cellular adhesion to the membrane, and further wherein thegrowth of the cells on the membrane provides enhanced cellulardifferentiation of the cells as compared to growth on the polymermembrane in the absence of the grooves and/or the pegs.

[0010] Specifically contemplated are microtextured polymer membranehaving projections may be about 1 μm, about 2 μm, about 3 μm, about 4μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm,about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about22 μm, about 24 μm, about 26 μm, about 28 μm, about 30 μm, about 32 μm,about 34 μm, about 36 μm, about 38 μm, about 40 μm, about 42 μm, about44 μm, about 46 μm, about 48 μm, about 50 μm, about 52 μm, about 54 μm,about 56 μm, about 58 μm, about 60 μm, about 62 μm, about 64 μm, about66 μm, about 68 μm, about 70 μm, about 72 μm, about 74 μm, about 76 μm,about 78 μm, about 80 μm, about 82 μm, about 84 μm, about 86 μm, about88 μm, about 90 μm, about 92 μm, about 94 μm, about 96 μm, about 98 μm,and about 100 μm. “Size” encompasses any of the dimensions of aprojection including the height (or depth in the event that theprojection is an inverted projection) of the projection from the baseplanar level of the membrane, the diameter of the projection or thewidth of the projection. The length of the projection may be 5 cm(50,000 μm) or more and may span the entire length of the wafer. Anycombination of these sizes for a given projection can have anycombination of the measurements of the exemplary values listed above.

[0011] The preferred size employed in any given culture system willdepend on the cell type being grown on the membrane. For example, forthe growth of fibroblasts, one of skill in the art would find itdesirable to employ membranes with projections in the lower end of therange, such as for example projections of about 2 μM to about 5 μM insize. For adult cardiac cells, one would preferably select membranesthat have mid-sized projections such as e.g., about 50 μM in size.Membranes having larger projections e.g., 100 μM in size would be betterfor growing adult skeletal muscle cells. Of course, these measurementsand cells sizes are merely exemplary and those of skill in the art willunderstand that the length and diameter of cell types can vary from afew microns to several hundred microns in size. Given the method andcompositions described herein, those of skill in the art should be ableto produce and employ the membranes of the invention to fit varyingcells types and sizes. Also it should be noted that not only the sizebut the spacing of the projections also may be varied.

[0012] A variety of shapes and forms are intended to be encompassed bythe term “projection.” Such a projection may be one which protrudes outof and above the surface of the membrane. Alternatively, a projectionmay be one that is configured inwards from the surface of the membraneso as to produce a dimple or indentation in the membrane. The shape ofthe projection may be regular or irregular and the projections may beregularly or irregularly positioned on the surface of the membrane. Theshape of the projections may be for example, conical, pyramid shaped,cylindrical, globular, rectangular or may be a heterogeneous mix ofshapes. The membrane may be arranged and shaped in any format commonlyused for culturing cells or indeed any shape that may be conducive toallowing a particular cell culture to grow in mass that mimics its invivo organ growth. The membrane may be planar, tubular, spherical,configured as a disk or stacked.

[0013] The membranes of the present invention are biocompatible. Theterm biocompatible as used herein generally refers to membranes whichare non-toxic, chemically inert, and substantially non-immunogenic whenused internally in the patient and which are substantially insoluble inblood or other bodily fluids. The term biocompatible is one that isgenerally understood by those of skill in the art and has been definedby the National Institutes of Health to encompass any substance that maybe placed in intimate contact with biological components without harmfuleffects. In addition to being biocompatible, the membranes of thepresent invention also have the desirable property of being deformable.The term “deformable” as used herein is intended to mean that themembranes have the ability to be mechanically deformed without loss ofintegrity of the surface microtopography or the surface chemicalmodification. The deformable membrane is such that it can withstand thephysiological range of stress/strain that the cells being cultured onthe membrane experience in vivo. For example, a heart cell experiencesextension of length changes that stretch the cell to +/−20% of itsresting size. Additionally the cell experiences a number of beats/minuteand a pulsatile pressure from blood flow. A membrane for growingmyocytes should preferably be able to withstand the application of suchextreme forces. On the other hand, bone cells for bone regenerationcannot withstand such pressures and/or forces and the membranes for thegrowth of such cells need not be as resilient as those used for growthof myocytes.

[0014] The chemical modifications of the membranes are resistant todeterioration upon application of mechanical stress. By “resistant todeterioration,” it is meant that the surface modification does notreadily fall off or become detached, degrade, undergo slippage, becomeremoved or otherwise be cleaved from the surface of the membranes of thepresent invention as compared to other non-deformable membranes.

[0015] The polymer material may be any polymer conventionally employedfor cell culture and may be for example selected from the groupconsisting of silicone, or other elastomeric polymers, hydrogels,biodegradables, bioerodible. Surface modifications contemplated to beuseful are those that allow for attachment of cells to the surface ofthe membrane, for example, through providing ligands for receptors thatmay be present in the cell surface of the cell to be cultured. Theinvention particularly contemplates surface modifications, whichcomprise attachment of laminin or fibronectin to the membrane, orpartial peptide sequences of laminin or fibronectin or modification oflaminin or fibronectin which nevertheless allow the laminin orfibronectin to act as a surface modification for the attachment ofcells. Growth of the muscle cells on the membranes of the presentinvention produces muscle cells that have contractile function and/orthe cells have mechanical deformation properties that are similar to themechanical deformation properties of said cells in vivo.

[0016] Also provided is a cell culture model for the growth anddevelopment of muscle cells comprising a membrane for the growth ofcells comprising a microtextured polymer membrane having projections ofbetween about 1 μm to about 100 μm in size and longitudinal grooves;wherein the polymer membrane comprises a surface modification tofacilitate cellular adhesion to the membrane, wherein the membranecomprises surface microtopography to facilitate cellular orientation;and further wherein the growth of the cells on the membrane providesenhanced cellular differentiation of the cells as compared to growth onthe polymer membrane in the absence of the grooves and pegs.

[0017] Other aspects of the invention contemplate methods of growinge.g., muscle cells comprising contacting the cells with the membrane ofthe present invention, under media conditions suitable to facilitate thegrowth of the cell wherein growth of the cells on the membranereproduces the physiological micro-architecture of the cells. Moreparticularly, the cells may be muscle cells and even more particularly,the cells may be myocardial cells. It is contemplated that the muscleand other cells grown on the membrane are responsive to neurohormonalstimulation. In alternative embodiments, it is contemplated that themuscle cells grown on the membrane exhibit contractile function thatmimic the contractile function of the muscle cell in vivo.

[0018] Also provided is a method of organogenesis comprising providingcells; contacting the cells with the membrane of the present invention;growing the cell in culture to allow the formation of tissue. Inpreferred aspects the membrane is a biocompatible membrane. In specificembodiments it is contemplated that the cells may be selected from othercell groups such as skeletal muscle, smooth muscle, cardiac muscle,vascular endothelial cells, lymphatic endothelial cells, stem cells,endothelial cartilage, bone cells or other cell types stimulated bymechanical force or subject to contact inhibition.

[0019] Other aspects, features and advantages of the present inventionwill be apparent from the entirety of the application, including thedrawings and detailed description, and all such features are intended asaspects of the invention. Likewise, features of the invention describedherein can be recombined into additional embodiments that also areintended as aspects of the invention, irrespective of whether thecombination of features is specifically mentioned above as an aspect orembodiment of the invention. Also, only such limitations which aredescribed herein as critical to the invention should be viewed as such;variations of the invention lacking limitations which have not beendescribed herein as critical are intended as aspects of the invention.

[0020] In addition to the foregoing, the invention includes, as anadditional aspect, all embodiments of the invention narrower in scope inany way than the variations specifically mentioned above. Although theapplicant(s) invented the full scope of the claims appended hereto, theclaims appended hereto are not intended to encompass within their scopethe prior art work of others. Therefore, in the event that statutoryprior art within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

[0021] Also, it should be understood that the detailed descriptionpresented below, while providing preferred embodiments of the invention,is intended to be illustrative only since changes and modificationwithin the scope of the invention will be possible whilst stillproviding an embodiment that is within the spirit of the invention as awhole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following drawing forms part of the present specification andis included to further demonstrate aspects of the present invention. Theinvention may be better understood by reference to the drawing incombination with the detailed description of the specific embodimentspresented herein.

[0023]FIG. 1 shows micro-pegged silicone membrane of the presentinvention viewed with phase microscopy.

[0024]FIG. 2A shows cardiac myocyte cultures growing on a “pegged”silicone membrane coated with laminin. FIG. 2B shows cells grown on asilicone membrane without pegs.

[0025]FIG. 3 shows cardiac attachment to micro-pegs (P) and intercalateddisc. FIG. 3A: two myocytes end-to-end span the gap between two pegs.FIG. 3B: Another myocyte seen attaching to a 10 μm diameter micro-peg atone end and to a fibroblast (F) at the other end.

[0026]FIG. 4 shows a histogram of degree of cell attachment in whichattachment is measured as the binding of a cell to an actual pegcompared to a virtual one (flat membrane with pseudo-pegs superimposedover the image).

[0027]FIG. 5 shows a vertical view to show narrow myofibril layer in acardiac myocyte grown on conventional flat membrane.

[0028]FIG. 6 shows cell nucleus and myofibrillar architecture atmicro-pegs (P), and cell height. In FIG. 6A: the cells are seen withconfocal microscopy, as above. FIG. 6B shows a histogram to showincreased cell height of cell grown on pegged membranes.

[0029]FIG. 7A shows the reaction steps in the surface chemicalmodification of silicone membranes: blank silicone (top), APTES,maleimide and peptide (bottom). FIG. 7B shows I) C(1s) and II) N(1s)core level x-ray photoelectron spectra of blank silicone (A), APTESlayer on silicone (B), maleimide layer (C) and peptide layer (C).

[0030]FIG. 8 shows the results of flexing of the iodinated peptidemodified silicone membranes for 48 hrs under cell culture media. 79% ofthe covalently bound peptide (Maleimide) and 59% of the non-covalentlybound peptide (Blank) remain on the surface following flexing.

[0031]FIG. 9 shows the fibroblast cell count on blank, APTES, andpeptide modified (10 and 100 μM) silicone membranes. Cell count shownbefore (left) and after trypsinization. Only the peptide surfaces showhigh cell counts after trypsinization. The control is tissue culturepolystyrene control. 10 and 100 μM refer to the input concentration ofthe peptide solutions used to prepare the membranes.

[0032]FIG. 10A-FIG. 10D shows phage images of cancer cell lines grown on10 μm pegged silicone membranes. FIG. 10A shows Mel-1 cells. FIG. 10Bshows Mel-1 cells. FIG. 10C shows Mum-2 cells. FIG. 10D shows Mum-2cells.

[0033]FIG. 11A and FIG. 11B show fibroblast proliferation on 10 μMpegged and flat silicone membranes, respectively, as observed on day 5of growth.

[0034]FIG. 12 shows cardiac fibroblast cell proliferation on flat and 10μM pegged silicone membranes.

[0035]FIG. 13. Cyclin D1 protein expression in cardiac fibroblastscultured on flat and pegged silicone membranes. Cyclin D1 protein issignificantly reduced in cardiac fibroblasts cultured on peggedmembranes compared with flat. Actin protein, not shown, remainedunchanged.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present invention is directed to a novel cell culture systemto study the process of myocyte remodeling in vitro which maintains adifferentiated in vivo myocyte cell phenotype. The culture system of thepresent invention, created by microfabrication technology coupled withsurface chemistry, allows cells to be grown which more closely mimic invivo heart physiology. Further, the new culture system yields cellswhich are responsive to both mechanical and neurohormonal stimuli thatare operative in the intact, failing heart in vivo.

[0037] In one of the preferred aspects of the present invention,membranes are provided for the growth of muscle cells such that myocytesgrown on these membranes obtain the physiological micro-architecture ofmyocyte cells seen in vivo. In order to produce the membranes,microtextured pegs of varied height are used to generate a polymericmembrane to maximize perpendicular surface for attachment of myoctyes orother cell types thereby permitting force transmission to the myocytesor other cell types in culture that mimics the force transmissionexperienced by myocytes or other cell types in vivo. Secondly,microtextured grooves of varied length and depth are used to increasecell alignment and circumferential attachment resembling the costamericstructural composition for lateral force transmission to the musclecells in culture. Thirdly, microtextured pegs and grooves are used tosimultaneously promote attachment and alignment of myocytes on thepolymer membranes with the desired elastomeric, optical, chemical, andbiocompatible properties of such cells in vivo.

[0038] In addition to providing membranes that promote the appropriatephysiological micro-architecture of the myocytes in culture, the presentinvention further modifies the membranes to alter the surface chemistryof microtextured membrane to promote attachment, adhesion-dependent cellsignaling and growth of the cardiomyocytes in culture. Moreparticularly, receptor ligands are attached to the surface of themembranes to facilitate the cellular adhesion. In preferred embodiments,the fibronectin receptor ligand GRGDSP (SEQ ID NO:1) and/or the lamininreceptor ligand YIGSRC (SEQ ID NO:2) are covalently bound to thesurfaces of the membrane.

[0039] The novel, microtextured, adhesive membranes of the presentinvention allow aligned, anatomically correct adult-phenotype myocytesto form in vitro. Typically, these cardiac myocytes are more “muscular”and can be used to study cardiac adaptive and patho-physiologicalprocesses in vitro without the complexity introduced by whole animalsequella to altered cardiac output. The significance of this in vitromodel is that growth of fully functional cells on substrata is anessential step in the path towards heart organogenesis and cardiactissue engineering. Furthermore, the culture systems of the presentinvention can provide an in vitro model of cardiac cells in progressionto heart failure and for remedies and reversal of such undesirableoutcomes. While the present invention generally discusses myocytes inculture, it should be understood that the membranes of the presentinvention also will be useful for the study of mechanobiology of othercell types known to respond to load, such as, for example, bone,connective tissues, endothelial cells (e.g., vascular endothelial cellsand lymphatic endothelial cells), stem cells, smooth and skeletalmuscle. Of particular interest, the inventors have demonstrated thatcancer cells can be grown on the membranes of the present invention tomimic their in vivo, tumor growth behavior.

[0040] The membranes of the present invention provide a transparent,biocompatible surface with specific microarchitectures upon which cellsexhibit enhanced cellular adhesion due to increased surface area, threedimensional geometries that mimic the in vivo geometric myocyteenvironment and biocompatible attachment moieties. The microtopographyprovides anisotropic or directional growth for cells and thus canrecreate tissue architecture at the cellular and subcellular level.

[0041] Thus, production of fully functional myocytes is facilitated by athree-dimensional membrane which provides a greater surface area forprotein attachment, and consequently, for the adherence of cells beingcultured. As a result of the three-dimensionality of the membrane,muscle cells continue to actively grow, in contrast to cells inmonolayer cultures, which grow to confluence, exhibit contactinhibition, and cease to grow and divide. The three-dimensional membraneallows for a spatial distribution of cellular elements which is moreanalogous to that found in the counterpart tissue in vivo. The increasein potential volume for muscle cell growth in the three-dimensionalsystem may allow the establishment of localized microenvironmentsconducive to cellular maturation. It has been recognized thatmaintenance of a differentiated cellular phenotype requires not onlygrowth/differentiation factors but also the appropriate cellularinteractions. The present invention effectively recreates the tissuemicroenvironment. Details of the methods and compositions for the growthof muscle cells in vitro according to the present invention arepresented herein below.

[0042] I. Mycocardial Cells and Myocardial Contraction

[0043] To the extent that the present invention is directed, inpreferred embodiments, to the growth and differentiation ofcardiomyocytes in culture, the present section provides a discussion ofcardiac cell biology and the contractile properties of muscle cells.

[0044] When the heart grows bigger, individual myocytes get larger insize (Zak, Raven Press New York, 1984; Gerdes et al., Circulation 86,426-30, 1992) but probably not greater in number (Olivetti et al., NEngl J Med. 17;336(16):1131-41;1997). Thus, control of individual cellgrowth is an important factor for increasing the strength of the heart.An adult myocyte can hypertrophy in volume either by increasing thenumber of sarcomeres in length and/or the number of myofibrils in crosssectional area. The direction in which the cell grows has major clinicalconsequences for the mechanical output from the whole heart (Katz, RavenPress, New York 1992). In concentric hypertrophy, the heart wall isthick and cells have a large cross-sectional area while in eccentrichypertrophy the heart wall is thin and the cells are longer than normal(Anversa et al., Am J. Physiol. 243 :H856-H861.1982; Gerdes et al.,Circulation 86, 426-30, 1992). Although there is much descriptive dataon the ability of the cells to control their shape in response to load(Thompson et al., Circ. Res. 54:367-377, 1984; Cooper, Ann Rev. Physiol.49:501-518, 1987), little is known about the regulation of these growthprocesses.

[0045] Although there is a significant amount of information availableregarding the overall processes of transcription, translation andprotein degradation in hypertrophy, there has been a lack of adequateculture systems to study mechanical signal transduction and assembly ofcontractile units (myofibrils) up until the present invention have notbeen addressed. The inventors contemplate that mechanical strain andneurohormonal stimulation are the primary physiological signals thatcontrol myofibril assembly and the net protein accumulation thataccounts for cell shape changes. The cell culture system of the presentinvention provides the first mechanism for investigating the effects ofsuch stimulation on myocytes.

[0046] The myofibril is the biological unit of contraction. In muscle,assembly of the sarcomeric proteins into highly organized myofibrils isan ordered and complex process. Formation of the first myofibril(myofibrillogenesis) is the process by which sarcomeres are assembled bybundling of the thick and thin filaments (Epstein and Fischman, Science251, 1039-44, 1991). A future striated muscle starts by looking morelike a fibroblast or smooth muscle cell with actin stress cablesanchored at the membrane and interspersed with dense Z-bodies. Much ofthis information has been attained by employing immunochemistry or byintroduction of green fluorescent protein (GFP) labeled proteins thatcan be visualized in living cells (Sanger et al., J Cell Biol 102,2053-66, 1986; Dabiri et al., 1997). It is contemplated that suchtechniques will be useful in the present invention to determine thephysiological micro-architecture and contractile function of themyocytes grown according to the present invention.

[0047] Myofibrils in cultured cardiac myocytes form outwards from focaladhesions (Lin et al., J Cell Biol 108,2355-67, 1989; Schultheiss etal., J Cell Biol 110, 1159-72, 1990), where cells attach to theextracellular matrix via integrins. Terracio et al. (Circ. Res.68:734-744, 1991) first demonstrated the presence of integrins on thecell surface of freshly isolated adult, and cultured neonatal cardiacmuscle cells. These transmembrane cell surface receptors connect ECMcomponents (collagens, laminin, fibronectin) to cytoskeletal elementswithin the cytoplasm of individual myocytes.

[0048] In the intact adult muscle, the attachment sites are found aroundthe circumference at the Z-line (costameres) providing a direct link forthe transmission of mechanical forces externally to do useful work inpumping blood. In cultured cells, the integrins and attachment sitesre-form only at the cell-substratum interface. In both cases, internalforces are transmitted throughout the cytoskeleton and perhaps even tothe nucleus (Pardo et al, J Cell Biol 97:1081-1088, 1983). Cardiacmyocyte integrins are of the β1-type with several different α-subunits.These heterodimeric complexes provide intracellular binding sites forcytoskeletal proteins (vinculin, paxillin, tensin, talin, α-actinin,dystrophin, etc.) which are localized to the cytoplasmic face of thecostamere or focal adhesion. These non sarcomeric cytoskeletal proteinsthus physically link integrins to sarcomeric actin filaments thatterminate at or near these sites. In addition to their structural role,cardiac myocyte focal adhesions and costameres may also be major sitesof mechanochemical signal transduction during myocyte remodeling, astheir organization appears to be highly regulated by externally appliedor intrinsically generated mechanical load (Simpson et al., J Cell Biol123:323-336, 1993; Sharp et al., Am. J. Physiol, 42: H546-H556, 1997,Eble et al., Am J Physiol Heart Circ Physiol., 278(5):H1695-H1707,2000). The role of the costameres and focal adhesions during addition ofnew filaments to existing myofibrils in hypertrophying cardiomyocytes ispresently unclear, but may now be addressed given that the presentinvention for the first time provides cell culture systems for the studyof such a role.

[0049] There is a constant turnover of proteins of the contractile unitsin cardiac myocytes (Low et al., J Cell Biol 56, 590-5, 1973; Morkin etal., Biochim Biophys Acta 324, 420-9, 1973; Wikman-Coffelt et al., JBiol Chem 248, 5206-7, 1973; Koizumi et al., J Biochem (Tokyo), 76(2):p. 431-9, 1974; Zak et al., J Biol Chem. 252(10): p. 3430-5, 1977). Inorder to understand such replacement at the level of the contractilemachinery, contractile proteins have been labeled and followed(Eisenberg et al., J Mol Cell Cardiol. 23(3): p. 287-96, 1991; Russellet al., Am J Physiol 262, R339-45, 1992; Rhee et al., Cell MotilCytoskeleton 28, 1-24, 1994). Contractile proteins in vivo are among thelongest lived of known proteins. For example, the myosin heavy chain,MyHC, turns over with a half-life of 7-10 days, whereas sarcomericactin's half-life is approximately 20 days. Sarcomeric proteinhalf-lives vary with age, and are influenced by the hemodynamic loadplaced upon the muscle cell.

[0050] The effects of mechanical load on contractile protein synthesisand degradation have also been studied in vitro, despite shortcomings inthe model system of randomly oriented cardiomyocytes maintained in 2-Dculture. For instance, it has been demonstrated that inhibition ofcontractile activity by blockade of calcium transients or inhibition ofactin-myosin crossbridge cycling reduces the MyHC and actin content ofcultured cells, and leads to a time-dependent disappearance of intactsarcomeres. These effects are entirely reversible, and result from botha decrease in MyHC and actin synthesis, and an increase in the rate ofMyHC and actin degradation (Samarel et al., Am J Physiol 263, C642-52,1992; Sharp et al., Circ.Res. 73: 172-183,1993; Byron et al., Am JPhysiol 271, C01447-56, 1996). Furthermore, static stretch of randomlyoriented, 2-D cultures of neonatal myocytes partially suppressed theaccelerated degradation of sarcomeric proteins in contractile-arrestedcells (Simpson et al., Am J Physiol 270, C1075-87, 1996). Stretch alsocauses MyHC and actin accumulation in contracting cells, again due toboth an increase in the rate of protein synthesis and a reduction in therate of degradation.

[0051] The signal transduction pathways responsible for load-inducedalterations in contractile protein synthesis and turnover are not known,but are the subject of current, intense investigation. Interest in theseprocesses relates to the fact that abnormal growth and remodeling ofcardiac muscle accompanies many common cardiac diseases, and is anindependent risk factor for cardiac morbidity and mortality.Nevertheless, understanding of these highly regulated events remainslimited, due to the lack of physiologically relevant cell culture modelswherein mechanical loading is applied to properly oriented, 3-D cultureswith appropriate ECM-cell attachments. The present invention providessuch a culture system for the first time. Using this system it is nowpossible to mechanically deform cardiac cells attached onchemically-bonded, microtextured surfaces prepared by the presentinvention in order to observe the morphology, growth and gene expressionin static versus cyclic stretched myocytes on microtextured membranes.

[0052] II. Microfabrication of Membranes

[0053] The techniques of microfabrication and micromachining have beenrecently used to create precisely controlled biomaterial surfaces viaphotopatterning and etching (Desai et al., Biotechnol Bioeng 57:118-120,1998; Bhatia et al, Biotech. Prog. 14:378-387, 1998; Chen et al.,Biotech Prog. 14:356-363, 1998). Microfabricated substrates can provideunique advantages over traditional biomaterials due to their ability tocontrol surface microarchitecture, topography, and feature size in thenanometer and micron size scale, and control of surface chemistry in aprecise manner through biochemical coupling or photopatterningprocesses. With the capability to design components spanning from themillimeter down to the nanometer range, few other engineeringtechnologies can so closely parallel the microdimensional scale ofliving cells and tissues.

[0054] Traditionally, microfabrication has only been applied tosemiconductor materials due to their oxidation and etching properties,using expensive microfabrication equipment. Recently, however,techniques to translate micromachined structures from inorganic toorganic polymeric materials have been introduced (Schmidt and von Recum,Biomaterials, 12: 385-389, 1991; Bucaro et al, IEEE ConferenceTransactions 0-7803-3869-3/97:217-219, 1997). This opens up uniqueopportunities in biological and tissue engineering applications. One ofthe challenges in tissue engineering is to find a more suitable methodfor the fabrication of scaffolds of defined architecture to guide cellgrowth and development and to understand what exact factors guide thatgrowth and development. Several polymer processing methods are currentlyused, including solvent casting, fiber bonding, and membrane lamination.The disadvantage of these techniques lies in the fact that architectureis achieved by altering solute or solvent concentration, thus making itdifficult to attain precise reproducible features in the micro- andnano-meter range.

[0055] The ability to spatially localize and control interactions ofcell types on polymeric materials presents an opportunity to engineerhierarchically and more physiologically correct tissue analogs formechanical, biochemical, and functional testing. The arrangement ofcells in more complex two and three dimensional arrangements hasbeneficial effects on cell differentiation, maintenance, and functionallongevity. For instance, MyHC is 12-15% of the total protein content ofthe neonatal myocardium in vivo, but only 4-6% in randomly oriented, 2Dcultures of spontaneously beating neonatal myocytes. MyHC contentdecreases even more in contractile-arrested cells. Particularly instudies involving translation of mechanical stimuli via substratecycling or stretching to cells, it is important to ensure cellularorientation and substrate attachment. The present invention providesmembrane substrata for facilitating this objective.

[0056] The membranes of the present invention provide a transparentbiocompatible surface with specific microarchitectures upon whichmyocytes can be grown. In an exemplary procedure the microtexturedmembranes are prepared using silicone membranes. Starting with a cleansilicon wafer, a 5 μm conformal layer of light sensitive photoresist(Michrochem SU8-5, Michrochem Corp., Newton, Mass.) is spun onto thewafer at 1500 RPM for 30 seconds and soft baked at 90° for 6 minutes. Aphotomask is used to define the pattern on to the photoresist layer uponexposure to UV light. Arrays of 10 by 10 by 10 micron (L×W×H) pegs (withspacing 30 μM center to center by 100 μM center to center) are thusphotolithographically defined. These dimensions correspond to celldimension, as myoctyes in culture are typically 50 microns in length and10-15 microns in diameter. The resulting photoresist structure isdeveloped and hard baked. Subsequently, the surface is spray coated ordipped into adhesion demoter and a thin layer of parylene is depositedon the photoresist/silicon substrate. The parylene deposition layer isapproximately 25 microns in thickness. The parylene layer forms aflexible mold for the elastomeric silicone. Subsequently, silicone(polydimethysiloxane), which is prepared by mixing elastomer andcatalyst (A103 Factor II Inc.) in a 10:1 ratio, is deposited on top ofthe parylene mold and allowed to cure at room temperature for 24-48hours. The silicone can then be peeled off the parylene and cut to thedesired shape and size.

[0057] The process for creating microgrooves is similar to the aboveprocess for creating micropegs except that a positive phostoresist isused. Shipley 1818 photoresist is spun on the wafer at 500 RPM for 180seconds. After a 5 minute soft bake the wafer is patterned with a maskaligner for 13 seconds at 20 mW. This results in longitudinal grooves of5 micron depth. The width and spacing of the grooves can be adjusted asdesired according to the mask. The wafer is placed in developer (351Shipley) for 0.9 minutes with continuous motion and rinsed withdeionized water. The purpose of the longitudinal grooves is to orientthe myocytes and also to provide a greater surface area for lateralattachment.

[0058] It should be understood that given the teachings of the presentinvention it will be possible for those of skill in the art to producearrays that correspond to dimensions smaller or larger than thoseexemplified here and still produce a membrane that will be useful forthe growth of cells that bear load.

[0059] As indicated elsewhere in the specification, most of theobservations to date presented have come from two-dimensional culturedmuscle. This is a limiting system in that the myofibrils can only makecostameres (attachments) on the bottom surface of the dish and lack thefascia adherens at the ends of the cells. Cultured myocytes at presentare (1) not oriented, (2) weakly adherent, and (3) notthree-dimensional. The myocytes lack an important third dimensionthrough which useful force is transmitted to the external worldsurrounding the cell. Early studies have shown that myocytes grow inmore physiological arrangements (i.e. muscle-like configurations) whenattached to perpendicular, rather than parallel, surfaces created by apin impaled in a soft dish (Yeoh and Holtzer, Experimental CellResearch, 104(1):63-78, 1977) or by Vandenburgh's less well knownhorizontal device (Vandenburgh et al., FASEB J. 5;2860-2867, 1991). Themethods for introducing microtopography into the membrane surfaces aspresented herein will overcome these architectural defects in cardiaccell anatomy and physiology.

[0060] The dimensions of the topographic features on which the cellsgrow will be modified to correspond to cell dimensions (typically 10-50μm size range). These platforms will provide a transparent biocompatiblesurface with specific micro-architectures upon which it is hypothesizedcells will exhibit enhanced cellular adhesion. The microtopographyprovides anisotropic or directional growth for cells and thus, canrecreate tissue architecture at the cellular and subcellular level in areproducible fashion.

[0061] For all experimental conditions in Example 2, unmodified (flat)and modified (textured) substrates are placed into culture dishes andseeded with appropriate cells. The effect of surface microarchitectureon cellular attachment and morphology is quantified by image analysis.Cells are fixed with 2% paraformaldehyde and proteins localized usingvarious antibodies (Terracio et al., In Vitro Cellular and DevelopmentalBiology. Vol. 24, 1988) to examine morphology under epifluorescentmicroscopy. Fixed samples also may be studied under SEM to observeinterfacial properties. Total protein, DNA and myosin content also maybe assessed by standard methods. Details of the immunochemistry andother methods are given in Example 1.

[0062] Use of Microtextured Pegs of Varied Height to MaximizePerpendicular Surface for Attachment Permitting Force Transmission as invivo

[0063] The perpendicular surface of the membrane may be optimized forcellular attachment. Immunolocalization can then be used to viewcontractile and focal adhesion proteins. Further, attachment may beassessed by cell density, total protein per DNA, and myosin to totalprotein ratios.

[0064] The silicone membranes with microtextured pegs of varied heightoptimize adherence and mimic a three-dimensional in vivo environment.Preferably, the membranes are thin (approx. 250 μm) with surfacetopologies consisting of small finger-like projections (pegs).Preferably, peg heights are 5 to 30 μm to cover the size range ofcardiac myocyte height in vivo.

[0065] A quantitative epifluorescent and phase light microscopy may beused to anatomically characterize the cells from cell culture; however,a confocal microscope is needed to provide the three-dimensionalstructure. Confocal microscopy enables one of skill in the art to viewthree planes for analysis: the conventional view from above the dish asthe X-Y plane, the longitudinal Z-Y plane, and the transverse Z-X plane.It has been previously shown that myofibrils only form on the bottomsurface of muscle cells in culture (Eisenberg, Am. J. Physiol.22;C349-C363, 1987). This can now be viewed with rhodamine phalloidin inconjunction with DAPI stain which serves to contrast the nucleus. Thenature of the cell's attachment in the perpendicular plane between thevertical peg and the myocyte also can be examined through thevisualization of focal adhesion proteins.

[0066] The degree of attachment of cells on microtextured surfaces andflat membranes is determined, and focal adhesions, cell shape, andmyofibrils may be viewed. Attachment is assessed by cell density, totalprotein per DNA, and myosin to total protein ratios. The morphologicparameters that can be measured are cell surface area, cell perimeter,maximum cell length, and position of cell with respect to the texture.The myofibril height above the bottom of the cell is an index of threedimensionality. In traditional flat surfaces, myofibrils of half microndiameter are stacked only a few cells high. The inventors believe thatthe peg height allows a significant increase in the stacking ofmyofibrils. Therefore, confocal microscopy is used to measure themyofibril height in the Z-axis both at the nuclear location and close tothe peg. The lateral attachment of myocytes to the pegs is assessed bycounting randomly selected areas for the % myocytes attached to a peg.This is compared with the % of cells on flat culture dishes attached tovirtual pegs drawn on the photographs after images are captured asdescribed in Example 2.

[0067] Use of Microtextured Grooves of Varied Depth to Increase CellAlignment and Circumferential Attachment Resembling the CostamericStructural Composition

[0068] It is necessary to have parallel aligned myocytes for themechanical experiments. The purpose of the longitudinal grooves is toorient the myocytes. Grooves should have a cross sectional area thatwill encompass the cell. A 10 μm×10 μm cross-section may be used howevera 20 μm×20 μm also may be used if the cells do not settle into thesmaller groove. The height of the groove can be changed according to theheight required using the same mask by deeper etching. A positivephotoresist is used to create micro-grooves in the silicon membrane. Theunmasked areas of a positive photoresist are preserved upon exposure tolight yielding the grooves of specified dimension in the spun-onphotoresist.

[0069] The next factor to be considered is how closely the groovesshould be spaced laterally. Cells in the animal are polarized, oriented,cylindrical shapes with diameters of 10-15 μm. They are closely packedwith intervening connective tissue of a few microns. If the spacebetween the grooves is more than 30 μm, the myocardial cells in betweenthe grooves become randomly oriented. This lateral spacing variable maybe determined efficiently using several masks of different groovecross-section area and of different lateral spacing. For example, one ofskill in the art can start with three different masks e.g., with 15 μm,20 μm, and 30 μm laterally. In this manner the influence of spacing onorientation may be determined. Groove length should exceed the 50 μmcell length and may be up to an inch or more.

[0070] The degree of attachment and alignment of cells on microtexturedand flat membranes can be determined. Alignment is determinedstereologically as described in the methods in Example 1. Confocalmicroscopy is used to observe costameric formations circumferentially inthe three-dimensional cell culture system as compared to two-dimensionalflat membrane. To do this, the inventors use the conventional view fromabove the dish as the X-Y plane, the longitudinal Z-Y plane, and thetransverse Z-X plane.

[0071] Use of a Combination of Microtextured Pegs and Grooves toSimultaneously Promote Attachment and Alignment on Polymer Membraneswith Desired Elastomeric, Optical, Chemical, and BiocompatibleProperties

[0072] Once optimal groove and peg dimensions for cellular attachmentand alignment have been determined, a combination of two masks is usedto create the pegs and grooves on the same membrane. This allowsexploration of the combined effect of attachment and orientation on cellgeometry and size.

[0073] In addition, several biomaterials may be used to see if there isany difference in cellular attachment with different materials. Threedifferent polymers are particularly contemplated: polydimethyl siloxane(siloxane), Polylactic/glycolic acid (PLA/PGA), andpolyhydroxyethlmethacrylate (PHEMA). These represent the followingpolymer classes: an elastomer, biodegradable polymer, and hydrogel,respectively. It should be understood that other elastomers,biodegradeable polymers and hydrogels also may be used in place ortogether with those exemplified herein. The use of biocompatiblematerials in microfabrication processing is an important step forapplication of this technology in biology.

[0074] Observations of the textured surfaces use phase microscopy.Microscopy is used to verify that the membranes produced are indeed thedesired patterns and texture specified. Profilometry is used to measurefeature height and depth.

[0075] The development of cell culture platforms based on novel threedimensional cellular arrangements as provided by the present inventionwill provide insight into cell-material interactions for the developmentof improved in vitro cell culture matrices for investigation intocellular mechanobiology. It is envisioned that the incorporation ofmicrotexturing in such a platform will further facilitate the co-cultureand maintenance of differentiated cell states.

[0076] Through its ability to achieve highly controlled microarchitectures on size scales relevant to living systems (from microns tonanometers), microfabrication technology offers unique opportunities toengineer new tissue models for the investigation of biologicalphenomena. Microfabricated constructs comprised of specific cell typesmay be preferred because of their greater relevance to physiologicaltissue. The ability to spatially localize and control interactions ofseveral cell types on polymeric materials (elastomers, and hydrogels)presents an opportunity to engineer hierarchically and morephysiologically correct tissue analogs. The arrangement of multiple celltypes in two- and three-dimensional arrangements has beneficial effectson cell differentiation, maintenance, and functional longevity.Additionally, microfabrication can be used not only to create morecomplex substrates, but also understand fundamental processes in livingsystems. It represents an important step in the merging of disciplinesto solve important problems at the interface of biology and engineering.Techniques that may be used to prepare membranes of the presentinvention include photolithography, diamond turning, diamond ruling andlaser machining. Such techniques are well known to those of skill in theart, see e.g., Marc Madou, Fundamentals of Microfabrication, CRC Press,Baton Rouge, which describes casting processes at page 127 et seq.,diamond tooling at page 351 et seq., and other techniques described inTable 7.7 therein at page 358-359. Those of skill in the art also arereferred to Polymer Processing and Structure Development (Wilkinson andRyan, Kluwer Academic Press), which describes various methods of polymerprocessing.

[0077] III. Modifying the Surface of the Micro-textured Membrane

[0078] Chemical bonding protocols which alter the surface chemistry ofmicrotextured silicone and other substrata that form the membranes ofthe present invention will promote attachment, adhesion-dependent cellsignaling and growth of cardiomyocytes in culture.

[0079] Neonatal rat cardiomyocytes readily attach to Type 1 collagen,laminin, and fibronectin-coated surfaces, but the noncovalent nature ofthe interaction between the ECM protein(s) and the supporting substrataare not ideal for the application of mechanical load. Upon large orrepeated mechanical strains, cells detach from the commerciallyavailable silicone substrata. Here, the inventors have used chemicalbonding techniques to covalently link adhesive peptides to the surfaceof flat and microtextured substrata as produced above. The effectivenessof these adhesive peptides in promoting cell attachment, growth anddifferentiation of the cardiomyocytes will be compared to flat andmicrotextured membranes coated with the parent ECM protein (i.e.fibronectin or laminin). The inventors propose to use the adhesiveproperties of two peptides derived from fibronectin and laminin, whichwill be covalently bonded to flat and microtextured elastic substrata.In addition, it should be noted that the microtextured membranes of theinvention also may be formed using other integrins and adhesive peptidessuch as for example.

[0080] The techniques for covalently bonding peptides to a siliconsurface can be performed by a variety of conventional methods usingknown coupling agents and known derivatization methods which are wellknown to those of skill in the art. This invention also relates to thecovalent coupling of such peptides to the microtextured membrane surfaceeither directly or via an appropriate linking or spacer group. U.S. Pat.No. 4,789,601, incorporated by reference in its entirety, describes apolyorganosiloxane composition having a biocompatible surface. Thesurface of the composition is treated with a primary amine or a peptide.This patent is incorporated herein by reference as teaching methods ofmodifying silicone surfaces.

[0081] U.S. Pat. No. 5,733,538, incorporated herein by reference,describes surface-modifying copolymers having cell adhesion properties.The surface modification techniques and polymers described therein alsomay be useful in conjunction with the present invention. Moreparticularly, the patent discusses a hemocompatible surface-modifyingadditive for modifying polyurethane or polyurethane urea substrates. Theadditive has a polyurethane or polyurethane urea hard block or analternative block which is miscible with the poly(urethane) orpoly(urethane-urea) base polymer, a polysiloxane hydrophobic soft block,an optional hydrophilic spacer and a peptide selected from the groupconsisting of Arg-Gly-Asp, X-Arg-Gly-Asp (SEQ ID NO:4), Arg-Gly-Asp-X(SEQ ID NO:5) and X-Arg-Gly-Asp-X′(SEQ ID NO:6), wherein X and X′ areamino acids.

[0082] In preferred aspects of the present invention, peptides orproteins are covalently bound to the silicone surface by the followingmultistep process. This process has been derived from, but altered inseveral ways, from that published previously (Ferguson et al.,Macromolecules 26: 5870-5875, 1993. Xiao et al., Langmuir, 14:5507-5516.)

[0083] A. A silicone membrane is chemically functionalized with OHgroups by exposure to a 13.6 MHz, 20 W, continuous radio frequencydischarge plasma of 0.8 mtorr of water for 4 minutes.

[0084] B. An amine group is attached to the silicone surface viareaction with a 5% (v/v) solution of 3-aminopropyltriethoxysilane(APTES) in ethanol for 1 hour at 60° C. under Ar atmosphere, then washedin ethanol.

[0085] C. A sulfo-maleimide cross-linker is attached to the amine groupvia reaction with a 0.2 mM solution of sulfo-SMCC (Pierce) prepared in aboric acid (0.2 M)/borax (0.05 M) buffer at pH of 7.5 for 30 minutes,then washed with the boric acid/borax buffer.

[0086] D. A cysteine-terminated peptide or protein is attached to themaleimide cross-linker by reaction with a 10-100 μM peptide solution in0.1 M degassed phosphate buffer (pH 6.6) containing 1.2% (v/v) Tween 80for 20 hours. The peptide functionalized silicone is then washed byexposure to 13.9 mM SDS for 20 hours at room temperature.

[0087] Step D was demonstrated using the 15-residue peptide,acetyl-CGGEGYGEGRGDSPG-amide (SEQ ID NO:3), but it can be performedusing any cysteine-terminated peptide or protein. For example, thelaminin derived peptide YIGSRC (SEQ ID NO:2) can also be bound in thisfashion, via a peptide tether. Mixtures of the two peptides can bereadily prepared from mixtures of their solutions. Polyethylene glycolcan be bound along with the peptides or proteins to the siliconesurface, to resist non-specific protein adsorption during cell growth.The entire process has been performed on both flat and microtexturedsilicone membranes. Those of skill in the art are referred to Lateef etal., which provides additional teachings of peptide-surface modification(Lateef et al., Amer. Chem. Soc. Polym. Mater. Sci. Engin. Prepr.85:403-404, 2001).

[0088] Selective removal of the amine produced by Step A. can beachieved by low energy argon ion bombardment of the membranes (Ada, etal., J. Vac. Sci. Technol. B 13: 2189-2196, 1995). Alternatively, an Arplasma treatment might also allow this amine removal. The selectiveremoval of amine prior to Step B will ensure that peptidefunctionalization will only occur on the walls of the microtexturedmembranes.

[0089] Analysis of the Cardiomyocyte Adhesive and Growth-promotingProperties of the Microtextured “Peg and Groove” Silicone Membranes inwhich GRGDSP (Fibronectin Receptor Ligand; SEQ ID NO:1) and the YIGSRC(Laminin Receptor Ligand; SEQ ID NO:2) are Covalently Bound to theirSurfaces

[0090] Cell attachment efficiency can be analyzed as previouslydescribed (Samarel and Engelmann, Am J Physiol 261, H1067-77, 1991).Briefly, plating efficiency is analyzed as the amount of recovered DNAfrom adherent cells 4 h after plating compared to the amount of DNA inthe plating suspension. In the case of Type I collagen-coated plasticdishes, plating efficiency of freshly isolated neonatal rat ventricularmyocytes was 68±4% (Samarel and Engelmann, Am J Physiol 261, H1067-77,1991). It is expected that plating efficiency will vary between flat andmicrotextured surfaces, and with the two peptides (whether used alone orin combination).

[0091] Adhesion-dependent Cell Signaling

[0092] Adhesion of cardiomyocytes to a flat, plastic substratum coatedwith Type I collagen increased the cellular content oftyrosine-phosphorylated proteins over time. Similar experiments can beconducted with flat and microtextured membranes containing covalentlybound GRGDSP (SEQ ID NO:1) and YIGSRC (SEQ ID NO:2). In addition,adhesion-dependent activation of growth-related signaling cascades canbe analyzed and the cellular content of tyrosine-phosphorylated FAK,paxillin and vinculin can be analyzed by immunoprecipitation withphosphotyrosine-specific antibody, and Western blotting with antibodiesspecific for the cytoskeletal proteins (Eble et al., In TheHypertrophied Heart, Takeda N, Dhalla NS, eds., Kluwer AcademicPublishers, Boston, 1999). The degree of adhesion-dependent activationof downstream MAPK cascades also may be assessed using quantitativeWestern blotting. Antibodies specific to the phosphorylated forms ofERK1/2, iNK and p38 MAP kinases may be usefully employed in thisendeavor. Activation of adhesion-dependent cell signaling will varybetween flat and microtextured surfaces, and with the two peptides(whether used alone or in combination).

[0093] Cell Growth and Differentiation

[0094] In addition to these “acute” adhesion studies, the effects of thedifferent microtextured, functionalized silicone membranes on specificgrowth parameters of the adherent cardiomyocytes can be examined. Totalprotein/DNA and α- and β-MyHC/DNA can be determined 48-72 h afterinitial plating using well-established techniques known in the art (Ebleet al., Am. J. Physiol. 274:C1226-C1237, 1998). These measurements canbe correlated with measurements of total cell volume and sarcomericassembly obtained by confocal microscopy. MyHC synthetic rate may bemeasured by [³H]leucine pulse-labeling, and MyHC protein half-lifeanalyzed in [³⁵5]methionine pulse-chase biosynthetic labeling (Samarelet al., Am J Physiol 263, C642-52, 1992). Previous studies from theinventors laboratories indicate that sarcomeric assembly andcardiomyocyte hypertrophy are closely correlated with both enhanced MyHCsynthesis and stability (Eble et al., Am J Physiol. 274:C1226-C1237,1998). MyHC synthesis and turnover thus serve as useful surrogatemarkers of cardiomyocyte growth and differentiation. In other words, 3-Dcardiomyocytes maintained on microtextured substrata with optimalconcentrations of functionalized peptides should demonstrate higherrates of MyHC synthesis and accumulation than their 2-D counterparts.Finally, the cellular content of FAK, paxillin, vinculin andβ1-integrins can be analyzed by quantitative Western blotting (Sharp etal., Am J Physiol, 42: H546-H556, 1997; Eble et al., In TheHypertrophied Heart, Takeda N, Dhalla NS, eds., Kluwer AcademicPublishers, Boston, 1999), and correlated with confocalimmunocytochemical studies of focal adhesion formation of cells grown onflat vs. microtextured substrata derivatized with the two integrinreceptor ligands. The amounts of these focal adhesion and cytoskeletalproteins will vary between flat and microtextured surfaces, and with thetwo peptides (whether used alone or in combination).

[0095] Myocyte Morphology in Response to Different Plating Substrates

[0096] Recent studies have revealed the importance of ECM-cellinteractions in myocyte attachment and spreading. In these studies,freshly isolated neonatal rat cardiac myocytes plated onto flat, plasticsurfaces coated with various ECM components demonstrate differentdegrees of cell attachment and spreading depending on the type of ECMprotein employed. Cells attach and spread to plastic surfaces coatedwith Type I collagen, Type IV collagen or fibronectin (Eble et al., Am JPhysiol Heart Circ Physiol., 278(5):H1695-H1707, 2000). However, lamininalone does not support cell attachment and spreading as well as theother ECM components tested, whereas poly-L-lysine provides a pooradhesive surface. These results are in keeping with the relativeabundance of collagen-, fibronectin- and laminin-specific β1-integrinson the cell surface of neonatal cardiomyocytes (Terracio et al., Circ.Res. 68:734-744, 1991).

[0097] Neonatal rat ventricular myocytes may be isolated and platedovernight at high density onto plastic dishes pre-coated with eithercollagen I (Col I), collagen IV (Col IV), fibronectin (FBN), laminin(LMN), or poly-L-lysine (Poly(Lys)). The myocytes are then maintained inserum-free culture medium for 48 hours. Hoffman modulation contrastmicrographs show that plates pre-coated with Col I, Col IV and FBN seemto be the best substrates for myocyte attachment and spreading. Myocytesplated on the LMN and Poly(Lys) pre-coated dishes attached less well andappeared less well spread.

[0098] Adhesion-dependent Increases in Phosphotyrosinylated Proteins inControl and Verapamil-treated Myocytes

[0099] The interaction of cardiac myocytes with an ECM substratum notonly provides a physical site for cell attachment and spreading, butalso initiates a series of cell signaling events leading to structuralreorganization and growth of the cardiac muscle cell. Protein tyrosinephosphorylation is one type of cell signaling event that occurs inresponse to integrin engagement and cell adhesion. A similar degree ofprotein tyrosine phosphorylation occurs in response to neurohormonalstimulation of the angiotensin II or endothelin receptor on the myocytecell surface. Cell attachment and spreading on a flat, collagen-coatedsubstratum induces the tyrosine phosphorylation of a variety ofintracellular proteins (Eble et al., Am J Physiol Heart Circ Physiol.,278(5):H1695-H1707, 2000). Many of these tyrosine-phosphorylatedproteins are localized in focal adhesions (Strait et al., Am J PhysiolHeart Circ Physiol., 280(2):H756-H766, 2001). However, tyrosinephosphorylation of cytoskeletal and other myocyte proteins is markedlyreduced in cells maintained in plating medium containing the L-type Cachannel blocker verapamil, a potent inhibitor of focal adhesion andcostamere formation in cardiomyocytes (Sharp et al., Am. J. Physiol, 42:H546-H556, 1997). This provides another method with which to monitor theeffects of cell attachment, spreading, and signaling on flat vs.microtextured substrata that are chemically modified with differentadhesive peptides.

[0100] Localization of Myosin Heavy Chain, Paxillin,Tyrosine-phosphorylated Proteins, and Focal Adhesion Kinase in NeonatalRat Ventricular Myocytes

[0101] Adhesion and growth of myocytes maintained on chemicallymodified, microtextured silicone and other substrata can also bemonitored by confocal immunolocalization of specific sarcomeric andcytoskeletal proteins (Deutsch et al., J Biomed Mater Res. AppliedBiomaterials, 53: 267-275, 2000). Cardiac myocytes plated onto plasticsubstratum coated with Type I collagen or laminin, display a typical“fried egg” appearance, with few, poorly organized sarcomeres asrevealed here by staining with a monoclonal antibody specific forsarcomeric MyHC. Focal adhesions, can be visualized with a monoclonalantibody specific for the cytoskeletal protein paxillin and are foundpredominantly at the bottom of the cell, and along the membraneperiphery. Focal adhesions are also the predominant sites for thelocalization of phosphotyrosinylated proteins, which include paxillin,vinculin and the nonreceptor protein tyrosine kinase FAK. Similarimmunocytochemical techniques can be used to monitor the distributionand amounts of focal adhesion proteins on microtextured as compared toflat substrata which have been chemically modified with integrin-bindingpeptides.

[0102] To monitor localization of contractile and signaling proteins inneonatal rat ventricular myocytes, cells may be cultured in serum-freemedium alone for 48 h, then fixed, permeabilized, and stained usingantibodies specific for sarcomeric MyHC; the cytoskeletal proteinpaxillin; proteins containing phosphorylated tyrosines; and FAK.

[0103] Using the above outlined studies, those of skill in the artshould be able to produce membranes that will promote myocyte adhesionand orientation, as well as produce cells with numerous focal adhesionsand costameres that are necessary for sarcomere assembly. The selectionof the optimal membrane microtopography can be based upon carefullydefined criteria which critically evaluate shape, structure and functionof the cells. The development of optically clear, microtexturedmembranes with covalently bonded adhesive peptides should provide thenecessary substratum with which to apply static or cyclic mechanicalload without the problems of cell detachment.

[0104] IV. Establishing a Mycocyte Cell Culture

[0105] The present invention can be employed in the in vitro growth ofany of a variety of cells including but not limited to myocardial cells,bone cells, connective tissues, endothelial cells, smooth and skeletalmuscle. The cells may be primary cells or may be cell lines derived fromsuch primary cells, tumors and the like. Cell lines derived from musclemay be obtained from a cell line depository such as for example AmericanType Culture Collection (ATCC, Bethesda, Md.). Such cell lines may besmooth muscle cell line, cardiac cell lines, skeletal muscle cells linesand the like. Further, the cell lines may be fibroblast cell lines thatare capable of differentiating into myocardial cells. The conditions forgrowth of the specific cell line purchased will depend on the biologicalsource and generally instructions for the growth of the cells are madeavailable along with the cell lines from ATCC.

[0106] Preferably, the cell lines are able to differentiate into cellsthat possess contractile function. Specifically preferred cells areembryonic or adult stem cells. The cells may be derived from anyvertebrate or non-vertebrate animal source. For example, the animalsource may be human, monkey or other primate, mouse, rat, rabbit, cat,dog, goat, sheep, pig, horse, cow, fish, bird or any other animal fromwhich such cells may be harvested. Preferrably, the cells for theculture in the present invention are mammalian cells. More preferably,the cells are human or primate cells, but rat and mouse cells also willbe usefully employed herein. Examples of cell lines and their culture onthe platforms of the present invention are detailed in the examplessection. The cells are inoculated onto the substrata. The appropriategrowth factors, may be added to the culture prior to, during orsubsequent to inoculation of the myocyte cells. The concentration ofsuch factors maintained in the cultures can be monitored and adjusted tooptimize growth.

[0107] Where the cells used are primary cells, they may be readilyisolated by disaggregating an appropriate organ or tissue which is toserve as the source of the cells being grown. This may be readilyaccomplished using techniques known to those skilled in the art. Forexample, the tissue or organ can be disaggregated mechanically and/ortreated with digestive enzymes and/or chelating agents that weaken theconnections between neighboring cells making it possible to disperse thetissue into a suspension of individual cells without appreciable cellbreakage. Enzymatic dissociation can be accomplished by mincing thetissue and treating the minced tissue with any of a number of digestiveenzymes either alone or in combination. These include but are notlimited to trypsin, chymotrypsin, collagenase, elastase, and/orhyaluronidase, Dnase, pronase, etc. Mechanical disruption can also beaccomplished by a number of methods including, but not limited to theuse of grinders, blenders, sieves, homogenizers, pressure cells, orsonicators to name but a few. For a review of tissue disaggregationtechniques, see Freshney, Culture of Animal Cells. A Manual of BasicTechnique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.

[0108] Once the tissue has been reduced to a suspension of individualcells, the suspension can be fractionated into subpopulations from whichthe myocyte and/or fibroblast cells can be obtained. This also may beaccomplished using standard techniques for cell separation including butnot limited to cloning and selection of specific cell types, selectivedestruction of unwanted cells (negative selection), separation basedupon differential cell agglutinability in the mixed population,freeze-thaw procedures, differential adherence properties of the cellsin the mixed population, filtration, conventional and zonalcentrifugation, centrifugal elutriation (counter-streamingcentrifugation), unit gravity separation, counter current distribution,electrophoresis and fluorescence-activated cell sorting. For a review ofclonal selection and cell separation techniques, see Freshney, Cultureof Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss, Inc.,New York, 1987, Ch. 11 and 12, pp. 137-168.

[0109] In specific examples, the isolation of myocytes may, for example,be carried out as follows: fresh muscle tissue is thoroughly washed andminced in an appropriate buffer in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin, collagenase or the like. The cellsare preplated onto uncoated plastic dishes to reduce non-muscle cellcontamination. The cells are plated at a relatively high density of, forexample, 1000-3000 cells/mm². Myocytes attach and spread overnight andmay be maintained in serum medium.

[0110] It is possible that the cells cultured in this manner may be usedfor transplantation or implantation in vivo. In such cases, it ispreferable to obtain the muscle cells from the patient's own tissues.After inoculation of the cells, the three-dimensional matrix should beincubated in an appropriate nutrient medium. Many commercially availablemedia such as DMEM, RPMI 1640. Fisher's Iscove's, McCoy's, and the likemay be suitable for use. It is important that the three-dimensionalmembrane be suspended or floated in the medium during the incubationperiod in order to maximize proliferative activity. In addition, theculture should be “fed” periodically to remove the spent media,depopulate released cells, and add fresh media.

[0111] These procedures are greatly facilitated when carried out using abioreactor, which is a closed system housing the three-dimensionalframework inoculated with muscle cells. A bioreactor reduces thepossibility of contamination, maintains the cultures under intermittentand periodic pressurization to create environmental conditions thatmaintain an adequate supply of nutrients to myocyte cells throughout thecartilage tissue construct by convection.

[0112] During the incubation period, the muscle cells will grow linearlyalong and envelop and colonize the three-dimensional membrane beforebeginning to grow into the openings of the matrix. It is preferable togrow the cells to an appropriate degree which reflects the amount ofmyocyte cells present in the in vivo tissue.

[0113] V. Uses of the Three-Dimensional Culture System

[0114] The three-dimensional culture system of the invention can be usedin a variety of applications. These include, but are not limited to,transplantation or implantation of either the cultured cells obtainedfrom the matrix, or the cultured matrix itself in vivo; screening theeffectiveness and cytotoxicity of compounds, allergens,growth/regulatory factors, pharmaceutical compounds, etc., in vitro;elucidating the mechanism of myocardial organogenesis; studying themechanism by which drugs and/or growth factors operate, to name but afew.

[0115] The growth of fully functional cells on the membranes of thepresent invention is a step in the path towards myocardial organogenesisand cardiac and other muscle tissue engineering. Three-dimensionaltissue culture implants may, according to the inventions, be used toreplace or augment existing tissue, to introduce new or altered tissue,or to join together biological tissues or structures.

[0116] The three-dimensional cultures may be used in vitro to screen awide variety of compounds, for effectiveness and cytotoxicity ofpharmaceutical agents, growth/regulatory factors, anti-hypertensiveagents, etc. To this end, the cultures are maintained in vitro andexposed to the compound to be tested. The activity of a cytotoxiccompound can be measured by its ability to damage or kill cells inculture. This may readily be assessed by vital staining techniques. Theeffect of growth/regulatory factors may be assessed by analyzing thecellular content of the matrix, e.g., by total cell counts, anddifferential cell counts. This may be accomplished using standardcytological and/or histological techniques including the use ofimmunocytochemical techniques employing antibodies that definetype-specific cellular antigens. The effect of various drugs on normalcells cultured in the three-dimensional system may be assessed.

[0117] The three-dimensional cultures of the invention may be used asmodel systems for the study of physiologic or pathologic conditions. Forexample, the new culture system may be used to determine the limits ofcell growth and mechanical signal transduction. Cardiac (high density,aligned, physiologically functional, micro-anatomically correct)myocytes grown on microtextured peg and groove membranes are maintainedin the unstretched state (control). Cells will then be mechanicallystimulated on the various microtextured and chemically bonded surfacesthat have been generated by the present invention. In this manner, newchemically modified, micro-textured surfaces will produce addition ofnew myofibrils reproducing the hypertrophy observed in response to bothphysiological and pathological stimuli. These experiments will helpprovide a better understanding of the mechanisms involved inpathogenesis of heart failure and normal adaption to exercise.

[0118] Those of skill in the art will understand that cultures grown onthe membranes of the present invention may have use as artificialorgan/tissue patch applications such as those described in, for example,a variety of U.S. Patents which are incorporated herein by reference.For example, U.S. Pat. No. 5,885,829 describes methods for regeneratingdental and oral tissues from viable cells using ex vivo culture on astructural matrix. The regenerated oral tissues and tissue-matrixpreparations thus provided have both clinical applications in dentistryand oral medicine. It is contemplated that the membranes and cellcultures of the present invention could similarly be employed toregenerate not only oral tissues but muscular, vascular and othertissue.

[0119] U.S. Pat. No. 5,721,131, incorporated herein by reference,describes a process for forming spatially oriented neo-vascularcapillaries. It is contemplated that the membranes of the presentinvention could be used in combination with the ultra-thin film patternof cell adhesion promoter and cell adhesion inhibitor wherein the celladhesion promoters have a line-width of between about 50-490 μm. Suchcompositions could be seeded on the present membranes and be used toallow allowing the endothelial cells to differentiate into spatiallyoriented neo-vascular capillaries.

[0120] U.S. Pat. No. 5,855,610 describes improved yields of engineeredtissue following implantation, and engineered tissue having enhancedmechanical strength and flexibility or pliability, can be obtained byimplantation, preferably subcutaneously, of a fibrous polymeric matrixfor a period of time sufficient to obtain ingrowth of fibrous tissueand/or blood vessels, which is then removed for subsequent implantationat the site where the implant is desired. The matrix is optionallyseeded prior to the first implantation, after ingrowth of the fibroustissue, or at the time of reimplantation. The method is particularlyuseful in making valves and tubular structures, especially heart valvesand blood vessels. It may be that the membranes of the present inventionalso may find use in such a method for engineering tissue. As such, U.S.Pat. No. 5,855,610 is incorporated by reference as teaching suchtechniques.

[0121] U.S. Pat. No. 5,804,178, incorporated herein by reference,describes a method of implanting a matrix structure having cellsattached thereto by providing a biocompatible polymeric matrix structurehaving attached thereto viable animal cells exhibiting normal growth andproliferation selected from the group consisting of endocrine cells,fibroblasts, endothelial cells, and genitourinary cells, which areallowed to attach thereto; and implanting the matrix structure havingcells attached thereto into a patient in need thereof, wherein thematrix structure is juxtaposed with tissue having high surface area andvasculature; adjacent the surface of the tissue selected from the groupconsisting of mesentery, omentum and peritoneum, and wherein the matrixstructure is configured to allow adequate nutrients and gas exchangebetween the attached cells and the blood for the cells to remain viableand to form tissue. The membranes of the present invention may similarlybe used to produce an implantable patch of cells for purposes of tissuehealing and/or regeneration.

[0122] In another example, U.S. Pat. No. 5,800,811, incorporated hereinby reference, describes an artificial skin prepared by impregnating acollagen matrix with a transforming growth factor-β having acollagen-binding site to bind the growth factor to the collagen matrix,incubating the impregnated matrix with a source of fibroblasts andmesenchymal stem cells to form a captured population of mesenchymal stemcells within the impregnated matrix and incubating the resultant matrixwith a source of keratinocytes which epithelialize the matrix to form anartificial skin. The membranes of the present invention could serve as auseful matrix in such techniques for generating artificial skin and assuch would be extremely useful in for example treating burns or otherskin tissue injuries.

[0123] Grafting cells into organs such as the brain also arecontemplated. Such techniques are described in e.g., U.S. Pat. No.5,750,103 in which a cellular graft is introduced into the brain of amammalian subject by attaching the cells to a support matrix so that thecell attaches to the matrix surface, and implanting the support matrixwith the attached cell into the brain. A membrane of the invention couldact as such an implantable support.

[0124] VI. Examples

[0125] The following examples present preferred embodiments andtechniques, but are not intended to be limiting. Those of skill in theart will, in light of the present disclosure, appreciate that manychanges can be made in the specific materials and methods which aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the invention.

EXAMPLE 1 Materials and Methods

[0126] The present example provides details of materials and methodsemployed throughout the application and in the Examples presented hereinbelow.

[0127] Polymeric Microtextured Membrane Preparation

[0128] Control silicone membranes are prepared using silicone membranes(Specialty Manufacture, MI) that are pre-treated with ION HCl for 2hours before coating with laminin to allow the cells to adhere. Themicrotextured polymeric membranes will be fabricated by a techniquewhere photolithographically defined silicon wafers are used as templatesor molds for reproducing complimentary images on desired polymers. Thisenables the reproduction of precise surface architectures andgeometries.

[0129] Silicone microtextured surfaces are produced using a methoddeveloped in the Desai laboratory as described herein throughout.Starting with a clean silicon wafer, approximately 1 ml of UV lightsensitive negatice photoresist is spun on the wafer for 30 seconds at1500 rpm. This results in PR thickness of approximately 10 microns. Thephotoresist is soft baked for 6 minutes at 95° C. The wafer islithographically patterned with arrays of 10 by 10 by 10 μm (L×W×H) pegsby exposure to 20 mW UV light for 10 seconds. It is then hard baked for4 minutes at 95° C. and then the pattern is developed. A parylene layeris deposited on the patterned photoresist and then peeled off, resultingin a parylene microtextured mold. Silicone gel (or any other polymericsystem) is prepared by mixing elastomer and catalyst in a 10:1 ratio andgently spreading over the parylene mold. After polymer curing (˜48 h)the microtextured membranes are subsequently coated with a thin layer oflaminin, or other bio-acceptable moiety.

[0130] Neonatal Rat Primary Cardiac Culture

[0131] Myocytes are isolated from the cardiac ventricles of 1-2-day oldSprague-Dawley rats by sequential collagenase digestion, as previouslydescribed (Samarel and Engelmann, Am J Physiol 261, H1067-77, 1991).Cells are pre-plated onto uncoated plastic dishes (60 mm) to reducenon-muscle cell contamination, and cells are plated at high density(1000-2000 cells/mm²) onto the various substrata (Goldspink et al., AmJ. Physiol. 271: H2584-H2590, 1996). Myocytes attach and spreadovernight, and are then maintained in serum-medium for 48 h. Cells aregrown on various silicone membranes at high density. All chemical andculture materials, unless otherwise specified, are obtained from SigmaChemical, St. Louis, Mo.

[0132] The Degree of Orientation

[0133] The images are selected at random. Orientation is measuredstereologically using digital images of the cells taken using e.g., anImagePoint 1.3. Images are overlaid by a neon green parallel lined grid.A count is taken of the number of intersections between the 0° lines andmyofibrils. Another count is also made of intersections between the 90°lines and myofibrils. To compute % orientation the following formula isused:

% orientation=(Iα−i/(α+I)×100,

[0134] where α=90° intersections and I=0° intersections

[0135] Image Analysis

[0136] Samples for each experiment are coded to remove subjective bias.At high magnification light microscopy, selection of alternate fieldsyields at least 30 cells per experiment. Five separate tissue cultureexperiments are analyzed for each condition. Images of cells closest tothe center of the alternating fields across the coverslip are captureddigitally in the Russell lab using a peltier cooled CCD video camera(Photometrics Image Point camera, Photometrics Ltd, Tucson, Ariz.).Image processing and analysis is performed using the Image-Pro Plussystem software version 3.0.01 for 95/NT (Media Cybernetics, SilverSpring, Md.) or similar software. The intensity (grey scale values)along a calibrated line for each cell is measured.

[0137] Confocal Measures

[0138] The Zeiss LSM 510 confocal microscope has computer graphics thatenable the cell to be treated in 3D space at 0.1 μm intervals. Cells areselected in a systematic random manner to provide statistically validsamples from at least five different cell cultures (Perhonen et al., JMol Cell Cardiol.30: 1713-1722, 1998). Total myocyte cell and myofibrilheights are assessed by scanning from the bottom to the top of the cellat three locations: (1) the center of the nucleus, (2) 15 μm from theedge of the nucleus where cells grown on flat membranes are 30% of theirmaximum height (Goldspink et al., J Cell Sci 110: 2969-2978, 1997), and(3) near the peg for pegged membranes. The frequency of peg attachmentby a myocyte is measured with phase microscopy or conventionalepi-fluorescence. Attachment is measured as the binding of a cell to anactual peg compared to a virtual one (flat membrane with pseudo-pegssuperimposed over the image). This is an important distinction as itcorrects for random occurrences. All the cells within a 160×240 μm² areaare used to analyze attachment to the peg.

[0139] Immuno-chemistry of Contractile, Focal Adhesion and CostamericProteins

[0140] Cells are fixed (15 min, room temperature) with 2% (w/v)paraformaldehyde in PBS, washed (15 min) in 1% (w/v) glycine in PBS, andpermeabilized (15 mm) with 0.5% (v/v) Triton X-100 in PBS. Myocytes arethen stained with commercially available antibodies to Myl-IC, paxillin,phosphotyrosine, vinculin, β1 integrin, and FAK. Appropriate FITC orrhodamine conjugated secondary antibodies are used to visualize thespecific proteins. Fluorescently labeled cells are then viewed using aZeiss Model LSM 410 or 510 laser scanning confocal microscope. Multipleoptical sections approximately 1 μM thick are taken of each sample toeliminate out-of-focus fluorescence of the intensely stained myocytes.

[0141] Biochemical Composition of Cultured Neonatal Rat VentricularMyocytes

[0142] For the quantitative analysis of total cellular protein and DNAcontent, cells are washed twice in HBSS, and 0.2N perchloric acid (1 ml)is added. The precipitated macromolecules are then quantitativelyscraped from the dishes and collected by centrifugation (10,000 g, 10min). The precipitate is redissolved by incubation (60° C., 20 min) in250 μl of 0.3N KOH. Aliquots are then used for analysis of total proteinby the Lowry method using crystalline human serum albumin as standard,and for DNA using 33258 Hoecht dye and salmon sperm DNA as standard, aspreviously described (Samarel and Engelmann, Am J Physiol 261, H1067-77,1991). For quantitative analysis of α-MyHC and β-MyHC content, cells arewashed twice in HBSS and lysed in 250 ml of sample buffer [62.5 mMTris-HCl, pH 6.8, containing 8% (w/v) of sodium dodecyl sulphate (SDS),5% (v/v) 2-mercaptoethanol, and 10% (w/v) glycerol]. The concentrationsof α-MyHC and β-MyHC isoenzymes are assessed by SDS-polyacrylamide gelelectrophoresis and silver staining (Samarel and Engelmann, Am J Physiol261, H1067-77, 1991). MyHC band intensity is quantified by laserdensitometry, and compared to the band intensity of purified MyHCstandards (0-300 ng). The positions of the α-MyHC and β-MyHC bands areconfirmed by electrophoresis of α-MyHC and β-MyHC protein standardsobtained from normal and hypothyroid adult rat hearts, respectively; andby Western blotting with an anti-MyHC antibody that cross reacts equallywith both isoenzymes.

[0143] Immuno Precipitation and Western Blotting for Analysis ofAdhesion-dependent Cell Signaling

[0144] Neonatal rat ventricular myocytes are rinsed with cold PBS andthen scraped in ice-cold lysis buffer according to Schlaepfer and Hunter(Schlaepfer and Hunter, 1996) 150 mM Hepes, pH 7.4 containing 150 mMsodium chloride, 10% glycerol, 1.5 mM magnesium chloride, 1 mM EGTA, 1mM sodium vanadate, 1 mM sodium pyrophosphate, 100 mM sodium fluoride,1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 10 μg/ml leupeptin,10 mg/ml aprotinin and 1 mM Pefabloc (AEBSF)]. Protein concentrationsare assessed using a bicinchoninic acid assay (Pierce, Rockford, Ill.)and then equal amounts of protein are immunoprecipitated with anti-FAK,paxillin, vinculin, or phosphotyrosine antibodies. Immune complexes arecollected by incubation with Protein A plus protein G agarose beads orprotein A or G beads alone for 2 hours at 4° C. The beads arecentrifuged, washed in Triton-only lysis buffer (lysis buffer withoutsodium deoxycholate and SDS), and then in a Hepes buffer containing onlysodium chloride, Triton X-100, and glycerol. The beads are thenresuspended in 8% SDS sample buffer and boiled to release the proteins.Proteins are separated by 7.5% SDS-PAGE and transferred tonitrocellulose membranes (Hybond, Amersham, Arlington Heights, Ill.).Blots containing anti-phosphotyrosine immunoprecipitates are probed withanti-FAK, vinculin or anti-paxillin antibodies. The blots containingFAK, vinculin, or paxillin immunoprecipitates are probed with ananti-phosphotyrosine antibody. Horseradish peroxidase-conjugated goatanti-mouse or goat anti-rabbit secondary antibodies are visualized byenhanced chemiluminescence (ECL, Amersham, Arlington Heights, Ill.). Thebands corresponding to FAK, or paxillin were quantified by laserdensitometry.

[0145] Application of Extrinsic Mechanical Load

[0146] Cyclic mechanical deformation is produced with a FlexercellStrain Unit (Model FX-3000, Flexercell International, McKeesport. Pa.),at varying cycles per mm and maximal strain for up to 48-72 h (Cadre etal., J. Mol. Cell. Cardiol. 30;2247-2259, 1998). In brief, theFlexercell Strain Unit consists of a vacuum manifold regulated bysolenoid valves that is controlled by a personal computer. The bottomsof the culture dishes are inserted into an airtight, sealed diaphragmatop the vacuum manifold and the entire apparatus is placed inside ahumidified CO₂ incubator. When vacuum is applied to the bottoms of theculture plates, the membrane bottoms are stretched to a user-definedpercentage of elongation (% strain). Varying patterns of strain (e.g.sinusoidal, stepwise, sustained, etc.) can be readily programmed usingfactory-installed protocols.

[0147] MAPK Activation

[0148] Different methods may be used to assess ERK1/ERK2 activation incardiomyocytes, the mobility shift Western blot, the “in-the-gel-kinase”(ITKA) assay, and quantitative Western blotting of cell extracts using aphosphospecific ERK1/ERK2 antibody (Promega). Methodological details ofthe mobility shift and 1TKA are found in Sabri et al., (1998a). Animmune complex assay with myelin basic protein as substrate also may beused, if necessary, to provide better quantitative results. JNK andp38MAPK activation are assessed by quantitative Western blotting withphospho-specific MAPK antibodies (Promega and New England Biolabs,respectively).

[0149] [³H]leucine Biosynthetic Labeling

[0150] Pulse biosynthetic labeling experiments are performed to assessMyHC fractional synthetic rates as previously described (Samarel et al.,Am J Physiol 263, C642-52, 1992; Sharp et al., Circ.Res. 73:172-183,1993). MyHC fractional synthetic rates (Ks, %/h) are estimatedfrom the following formula:

Ks=100[P*/(F*.t)]

[0151] where P* and F* are the leucine specific radioactivities in MyHCprotein and medium, respectively, and t is the labeling time in hours.Pulse-chase biosynthetic labeling. MyHC degradation is assessed inpulse-chase biosynthetic labeling experiments, as previously described(Samarel et al., Am J Physiol 263, C642-52, 1992; Sharp et al, Circ.Res.73: 172-183,1993; Eble et al., Am. J. Physiol. 274:C1226-C1237, 1998).Cells are incubated (24 h, 37° C.) in myocyte growth medium supplementedwith 8 μCi/ml of [³⁵S]methionine. At the end of the pulse-labelingperiod, cells are rapidly rinsed twice in HBSS and either harvested byaddition of 500 ml of SDS sample buffer, or chased for 24 h in growthmedium supplemented with 2 mM unlabeled methionine. Cell samples arethen separated by SDS-PAGE on 180 mm long, 0.7 mm thick, 7-17% verticalgradient SDS-PAGE gel. In each experiment, a constant fraction of thetotal protein of each culture dish is applied to individual gel lanes.This ensures that for all pulse-chase experiments, the amount ofradioactivity in MyHC declines by decay rather than by simple dilution.After electrophoresis, gels are autoradiographed with fluorographicenhancement. Dried gels are exposed to unflashed Kodak XAR-5 film forvarying time periods (24 days) at −80° C. Individual MyHC bands on theautoradiographs are scanned three times, and the average area beneaththe MyHC peak is computed by autointegration. Linearity of detection ofradioactivity by fluorography is assessed as previously described(Samarel et al., Am J Physiol 263, C642-52, 1992). The fractional rateof MyHC degradation (MyHC Kd, % Ihour) for each condition is estimatedby the following formula:

MyHC Kd=100[ln(MyHC AU)₀−ln(MyHC AU)₂₄]/24

[0152] where ln(MyHC AU)₀ and ln(MyHC AU)₂₄ are the natural logarithmsof the average absorbance (in arbitrary absorbance units) of the MyHCbands at times 0 and 24 hours of the chase. MyHC Kd values are thenconverted to apparent half-lives (in hours) according to the followingformula:

MyHCt _(½)=100[In(MyHC AU) ₀−ln(MyHC AU)₂₄]/24

EXAMPLE 2 Use of Surface Microtopography to Determine Cell Attachmentand Shape

[0153] Microtextured membranes are created using photolithography andmicrofabrication techniques described herein above. FIG. 1 showsmicro-pegged silicone membrane of the present invention viewed withphase microscopy. This membrane has rows of micro-pegs, each 10 μm highspaced 30 μm center to center along the row with 100 μm between the rows(center to center).

[0154] Primary rat neonatal cardiac myocytes were plated on laminincoated flat silicone membranes or those with micro-pegs 10 μm high toallow for perpendicular attachment. The changes in morphology wereassessed by comparing the frequency of peg attachment and cell heightand this reveals excellent myocyte shape and peg adhesion. FIG. 2A showscardiac myocyte cultures growing on a “pegged” silicone membrane coatedwith laminin. This membrane has rows of micro-pegs, each 10 μm high(seen as rows of bright circles) spaced 30 μm center to center along therow and 100 μm between the rows, center to center. Note that the cardiacmyocytes frequently terminate with a blunt end on a peg (arrows). Thereis also a tendency for the cells to straddle between the rows giving anoriented appearance. FIG. 2B shows cells grown on a silicone membranewithout pegs. These traditionally grown myocytes are randomly orientedand have tapered rather than blunt ends. The randomly dispersed circularblobs (asterisk) are tissue debris. FIG. 3 shows cardiac attachment tomicro-pegs (P) and intercalated disc. Horizontal views are seen withconfocal microscopy where myofibrils are light in this culture (actinseen by phalloidin staining). FIG. 3A: two myocytes end-to-end span thegap between two pegs. The cells are connected by an intercalated disc,rarely seen in conventional tissue culture. FIG. 3B: Another myocyteseen attaching to a 10 μm diameter micro-peg at one end and to afibroblast (F) at the other end. Note that the myocyte ends with acircular attachment to a peg (P) and the striated myofibrils (light,actin seen with phalloidin stain) lie in parallel bundles throughout thecell. FIG. 4 shows a histogram of degree of cell attachment in whichattachment is measured as the binding of a cell to an actual pegcompared to a virtual one (flat membrane with pseudo-pegs superimposedover the image). All the cells within a 160×240 μm² area from 3different cultures were used to analyze attachment to the peg. Cellsplated on pegged membranes attach more often (89.6±1.2%; n=3) to anactual peg than cells attaching to a virtual peg (15.4±3.0%; P<0.0001).

[0155] Myofibrils only form on the bottom surface of muscle cells inculture using electron microscopy (Eisenberg, Am. J. Physiol.22;C349-C363, 1987). This myofibril layer can now be rapidly viewed withimage analysis of confocal serial sections below by rotating the Z-stackinto the Z-Y plane. FIG. 5 shows a vertical view to show narrowmyofibril layer in a cardiac myocyte grown on conventional flatmembrane. Note below that the striated myofibrils (red, phalloidinstain) lie in parallel bundles close to the bottom of the cell and belowthe nucleus, N, (purple, DAPI stain), giving the abnormal appearance ofa “fried egg.” FIG. 6 shows cell nucleus and myofibrillar architectureat micro-pegs (P), and cell height. In FIG. 6A: the cells are seen withconfocal microscopy, as above. Note that the myofibrils reach to the endof the cell instead of the tapering into stress cables seen intraditionally cultured cells. In 3D rotation of this image themyofibrils enclose the nucleus. The cell has the a more life-likecylindrical shape. FIG. 6B shows a histogram to show increased cellheight of cell grown on pegged membranes. Confocal microscopy was usedto measure the total height of the cell. Cells plated on peggedmembranes are 42.9±2.1% (n=2) higher than cells grown on flat membranes(P=0.03).

[0156] The inventors conclude that the 3D topography of the surfaceaffects cardiac myocyte architecture and that cells prefer to terminateon a vertical structure with a subsequent increase in cell height.

EXAMPLE 3 Altering the Surface Chemistry of the Microtextured Membranes

[0157] This Example deals with chemical bonding protocols which alterthe surface chemistry of microtextured silicone and other substrata topromote attachment, adhesion-dependent cell signaling and growth ofcardiomyocytes in culture.

[0158] Covalent attachment of peptides to the silicone surfaces areused, as described above and depicted schematically in FIG. 7A. TheGRGDSP (SEQ ID NO:1) peptide sequence is known to activate the integrinbinding mechanism of various cell lines (Xiao et al., Langmuir 14:5507-5516, 1998). The peptide functionalized silicone membranes werecharacterized by radiolabelling and x-ray photoelectron spectroscopy.¹²⁵I radiolabelling of the peptide was performed, then the peptide wasbound to the silicone surface. FIG. 7B shows the I) C(1s) and II) N(1s)core level x-ray photoelectron spectra for the silicone surfaces atvarious stages of preparation (letters on spectra correspond to those inFIG. 7A). The appearance of new C(1s) components and the shift in theN(1s) peaks are both consistent with the chemistry depicted in FIG. 7A.

[0159] The ¹²⁵I-peptide functionalized silicone membranes were flexed ina Flexercell apparatus for 48 hours under cell growth media. The resultsof this experiments are shown in FIG. 8. Greater than 75% of thecovalently bound peptide (labeled “Maleimide”) remained bound to thesurface after flexing. By contrast, only about 60% of the noncovalentlybound peptide (labeled “Blank”) remains on the silicone surface aftersimilar flexing experiments. The binding of the peptide layer afterflexing in vitro is examined in the present Example because the firststep used by other researchers—oxygen plasma treatment (as opposed towater plasma treatment employed here)—leads to the formation of aloosely bound silica layer that is poorly coupled to the bulk silicone(Bowdin et al., Appl. Phys. Lett. 75: 2557-2559, 1999). It is generallyknown that silica films formed on polymers can crack or delaminate uponflexing (Yanaka et al., J. Appl. Phys. 90: 713-719, 2001). By contrast,the water plasma treatment utilized in the present invention simplymodifies the silicone with OH groups, rather than depositing a stiffsilica film. This ameliorates the problems of cracking and delaminationseen membranes which have silica films formed on polymers. Thus, themembranes described in the present invention are a significantimprovement over those available to those of skill in the art.

[0160]FIG. 9 shows the results of rat cardiac fibroblasts grown on thevarious chemically modified and blank silicone membranes. Both thepeptide concentrations (labeled “10 μM” and “100 μM” and the aminesurface (labeled “APTES”) demonstrated enhanced cell numbers compared tothe blank silicone after 24 hours. After trypsin washing, used to removebound fibroblasts, only the peptides displayed enhanced cell bindingcompared to both blank silicone and the tissue culture polystyrenecontrol. These results demonstrate that these peptide functionalizedsilicone surfaces enhance cell binding.

EXAMPLE 4 Dynamic Mechanical Pacing of Cardiac Myocytes

[0161] This example is directed to mechanically deforming cardiac cellsattached to traditional surfaces. The surface on which cells are growngreatly affects both the plating efficiency and the long-term attachmentduring mechanical manipulation. The best attachment of cardiomyocytes ison commercial plastic Petri dishes coated with Type I collagen (68±4%,n=6) but these are rigid so that cells cannot be moved on them. Thecollagen-coated elastic membranes that came with the original FlexercellFX-2000 System (Flex-1 plates) are acceptable (58±8% of plated cellsattach, n=10) but collagen-coated membranes provided with the newFX-3000 System (Bioflex plates) only allow a small and variable fractionof plated cells to adhere (38±12%, n=8). These initial numbers of cellsare even further reduced as soon as any mechanical manipulation isperformed on the Bioflex membranes. These results were particularlydisappointing, as the Flexercell FX3000 System provides a convenientmethod to subject cultured monolayers to either static or cyclic,uniform radial strain, an important factor that is not achievable withthe older FX-2000 System. Of course, neither commercially availablesystem provides microtextured membranes with chemically modifiedsurfaces to which ligand peptides are covalently attached. The manyshortcomings of these commercially available systems have thusstimulated the use of a variety of “home-made” stretching devices. Somedetails relevant to this application are briefly outlined below.

[0162] Static Stretch of Aligned Cultures

[0163] The lengths and widths of myocytes maintained at 10% stretch for6 hours are measured. Unstretched aligned cells grown on parallelstreaked collagen (Simpson et al., J Cell Physiol 161(1):89-105, 1994)are highly polarized with a length/width (L/W) ratio of 13.4±0.8compared to randomly-oriented controls where L/W is 4.2±0.4 (n=4,P<0.001). Aligned cells stretched 10% are even more polarized with L/Wratio of 22.6±2.9 (n=4, P<0.03). In addition, the cell nucleus isdistorted by stretching of the aligned cells with L/W nuclear ratioincreasing from 1.8±0.2 to 2.6±0.2 upon 10% stretch (n=4, P<0.02).Nuclei of randomly oriented cells are more circular (1.3±0.05).Therefore, it appears that stretch and alignment affect the shape ofboth the nucleus and the cell (Heidkamp and Russell, Cell TissueResearch, 305:1221-127, 2001).

[0164] Static Stretch of Randomly Oriented Neonatal Myocytes ProlongsMyofibrillar Protein Half-life

[0165] The inventors have demonstrated that intrinsic mechanical load inthe form of spontaneous contractile activity increases the rate ofmyofibrillar protein synthesis, and reduces the susceptibility ofcontractile proteins to intra-cellular proteolysis (Samarel andEngelmann, Am J. Physiol 261, H1067-77, 1991; Samarel et al., Am JPhysiol 263, C642-52, 1992; Sharp et al., Circ.Res. 73: 172-183,1993;Byron et al., Am J Physiol 271, C01447-56, 1996). The relationshipbetween external mechanical load (i.e. a 5% static stretch) andmyofibrillar protein degradative rates is explored in the same modelsystem (Simpson et al., Am J Physiol 270, C1075-87, 1996.) Spontaneouslycontracting, randomly oriented myocytes that were grown oncollagen-coated silastic membranes were maintained under controlconditions, or subjected to 5% linear stretch. Paired cultures weremaintained in serum-free medium containing nifedipine (12 μm) to inhibitspontaneous contractions. Myofibrillar structure was evaluated byconfocal and electron microscopy. Myofibrillar protein content anddegradation were assessed by SDS-PAGE and by pulse-chase biosyntheticlabeling experiments, respectively. Pulse-chase experiments revealedthat contractile arrest accelerated the loss of protein-bound tracerfrom the total myofibrillar fraction, and from pre-labeled MyHC andactin, but not desmin. Sarcomeric disassembly developed in parallel withthese metabolic changes. A 5% static load partially stabilizedmyofibrillar structure in nonbeating cells, and suppressed the loss ofisotopic tracer from the total myofibrillar fraction, MyHC and actin inboth beating and nonbeating cells. Contractile activity and/or staticstretch promoted the accumulation of MyHC, actin and desmin. Applying astatic load to myocytes that lacked pre-existing myofibrils did notpromote the assembly of sarcomeres or alter protein turnover. These dataindicate that rates of myofibrillar protein turnover are correlated withthe organizational state of the sarcomere, and that contractile proteinhalf-life can be prolonged by both intrinsic and extrinsic mechanicalload. Static stretch increases MyHC half-life in contracting andnifedipine arrested myocytes. Randomly oriented, spontaneouslycontracting cells grown on collagen-coated silastic membranes weresubjected to maintenance cultures, or 5% linear stretch. Pairedmembranes were maintained in medium containing nifedipine (12 μm) toinhibit spontaneous calcium transient and beating. MyHC half-life wasdetermined by pulse-chase biosynthetic labeling.

[0166] Cyclic Stretch Induces Myocyte Hypertrophy and Altered GeneExpression

[0167] The inventors examined whether extrinsic mechanical load in theform of cyclic stretch induced myocyte hypertrophy, and led todown-regulation of contractile and calcium handling genes which havebeen associated with the remodeled, failing cardiac myocyte in vivo(Cadre et al., J. Mol. Cell. Cardiol. 30;2247-2259, 1998). Randomlyaligned neonatal myocytes were maintained in serum-free culture mediumunder control conditions, or subjected to cyclic mechanical deformation(1.0 Hz, 20% maximal strain, 48 h) using the Flexercell FX-2000 System.Under these conditions, cyclic stretch induced hypertrophy, as evidencedby significant increases in total protein/DNA ratio, MyHC content, andANF secretion. A similar approach may be used in analyzing geneexpression changes associated with various mechanical deformations inthe new myocyte culture system.

[0168] Cyclic Stretch-induced Alterations in MyHC and ANF mRNAs

[0169] Neonatal rat ventricular myocyte cultures were maintained undercontrol conditions, or subject to cyclic stretch (48 h, 1 Hz, 20%maximal strain). Total RNA was isolated, size-fractioned, andtransferred to nylon membrane. Northern blots were sequentially probedwith ³²P-labeled oligodeoxynucleotide or cDNA probes specific for αMyHC,β MyHC, ANF and GAPDH mRNAs, and 18S rRNA). Probe binding was detectedby autoradiography, and quantified by scintillation spectroscopy.

[0170] Cyclic Stretch-induced FAK Phosphorylation

[0171] The signal transduction pathways that may be responsible forcyclic stretch-induced cardiac myocyte hypertrophy and altered geneexpression are also being investigated. In recent studies, it has beenfound that focal adhesion kinase (FAK) a nonreceptor protein tyrosinekinase localized to cardiac myocyte focal adhesions and costameres israpidly autophosphorylated in response to cyclic stretch. Focaladhesions may therefore serve to transmit mechanical deformations to thecell interior, as well as to provide a structural link between the ECMand the cardiac myocyte cytoskeleton. Of note, FAK activation has beenimplicated in both adhesion- and growth factor-induced cell signalingevents leading to proliferation, differentiation and cell survival inother cell types. FAK activation also can indicate an acute pressureoverload of the myocardium in vivo (Kuppuswamy et al., J Biol Chem14;272(7):4500-8, 1997), and is a critically important component ofendothelin-induced focal adhesion, costamere, and sarcomere assembly invitro (Eble et al., Am J Physiol Heart Circ Physiol.,278(5):H1695-H1707, 2000). Thus, these data indicate that mechanicaldeformation of cardiac myocytes elicits specific cell signaling eventsvia integrins and focal adhesion proteins that may be critical tocardiac myocyte growth and differentiation. Similar studies are plannedusing microtextured membranes with covalently bonded peptide ligands toassess the degree and time course of FAK activation as compared to cellsstretched on flat surfaces.

[0172] FAK phosphorylation is induced by cyclic stretch. Neonatal ratventricular myocytes cultured in serum-free medium on Flex 1 plates for48 h and then cyclically stretch (1 Hz, 30% maximal strain) for 2, 5,15, and 30 min using a Flexercell apparatus. Tyrosine phosphorylatedproteins were immunoprecipitated, size-fractionated by SDS-PAGE, andtransferred to nitrocellulose membrane. Blots were probed with ananti-FAK polyclonal antibody and the protein bands were visualized usingECL. Cyclic stretch induces rapid FAK phosphorylation by 2 min.

[0173] Endothelin-induced FAK Phosphorylation in Myocytes Plated onDifferent Substrates

[0174] Increased contractile activity (by endothelin) induces FAKphosphorylation. Neonatal rat ventricular myocytes were isolated andplated overnight at high density onto plastic dishes pre-coated witheither collagen I (Col I), collagen IV (Col IV), fibronectin (FBN),laminin (LMN), or poly-L-lysine showing morphology and adherence of thecells. Half of the myocytes were subsequently stimulated withendothelin-1 (ET-1, 100 nM, 5 min), a potent agonist that stimulatesfocal adhesion formation and sarcomeric assembly. Cell extracts werethen prepared from unstimulated [C] and ET-1 stimulated cells. Tyrosinephosphorylated proteins were immunoprecipatated, size-fractionated bySDS-PA GE, and transferred to nitrocellulose membrane. The resultingWestern blot was probed with an anti-FAK polyclonal antibody and theprotein bands were visualized using enhanced chemiluminescence. Note,ET-1 stimulated FAK phosphorylation in the myocytes plated on the Col I,Col IV and FBN pre-coated plates. FAK activation by ET-1 was lesspronounced in cells maintained on laminin. No ET-1 induced FAKphosphorylation was observed on poly-L-lysine coated dishes.

[0175] Focal adhesion, costamere and sarcomere assembly can also bestimulated in low-density, noncontracting cardiomycotes by treatmentwith various neurohumoral agents (e.g. angiotensin II, phenylephrine,ET-1) that induce cardiomyocyte hypertrophy and remodeling in vivo. Forinstance, both phenylephrine (50 μM) (Eble et al., Am. J. Physiol.274:C1226-C1237, 1998) and ET-1 (100 nM, 48 h) induce cardiomyocytehypertrophy, as evidenced by increased total protein/DNA and MyHC/DNAratios. Both agents also increase cell size, and stimulate the assemblyof newly synthesized myofibrillar proteins into sarcomeres (Eble et al.,Am. J. Physiol. 274:C1226-C1237, 1998; Eble et al., Am J Physiol HeartCirc Physiol., 278(5):H1695-H1707, 2000). Endothelin-induced sarcomereassembly is associated with an increase in focal adhesion and costamereformation, and also an increase in the localization ofphosphotyrosinated proteins into focal adhesions. Indeed, ET-1 causesthe rapid tyrosine phosphorylation of both FAK and paxillin (Eble etal., Am J Physiol Heart Circ Physiol., 278(5):H1695-H1707, 2000). Ofparticular interest is the intensity of both basal and agonist-inducedFAK activation varies depending upon which ECM component the myocytesare attached to. In this case, static or cyclic stretch, rather thanendothelin, is used to activate FAK. Different mechanical loadingconditions can be tested, as well as different adhesive peptides andmicrotextures.

[0176] The new culture system is used to determine the limits of cellgrowth and mechanical signal transduction. Cardiac (high density,aligned, anatomically correct, physiologically functional) myocytesgrown on microtextured peg and groove membranes will be maintained inthe unstretched state (control). Cells will then be mechanicallystimulated on the various microtextured and chemically bonded surfacesthat have been generated.

[0177] In order to stress the cells, the inventors use three classes ofmechanical distortions; namely (1) a single static stretch, (2) astaircase of small steps to mimic heart failure due to chronic increaseddiastolic volume, and (3) intermittent packets of activity to mimicexercise. The novel culture model provides the first opportunity tomimic physiological and patho-physiological conditions of the humanheart.

[0178] (1) Sudden overload. The inventors apply a sudden lengthextension of 5, 10 or 20% in the direction parallel (longitudinalstrain) or perpendicular (transverse strain) to the direction of themyofibrils. The inventors monitor rapid changes in phosphorylation ofspecific cell signaling proteins (FAK, paxillin, MAPK) over the timecourse from minutes to hours.

[0179] (2) Heart failure. The inventors use the dynamic strain system(Flexercell FX-3000) in order to deform the cells mechanically at agiven amplitude and frequency. In this aspect the inventors use multiplesmall stretches (steps) of varying length. Time between incrementalincreases in stretch will start at 5% every 2 h, and then be varied todetermine the amplitude and frequency of stepped increases that resultin maximal protein content (protein/DNA ratio). This will reproduce thepositive feedback circuit between load and mal-adaptation that plaguesthe failing heart.

[0180] (3) Exercise. Passive mechanical distortions to silasticmicrotextured membranes with a sine wave of 5% amplitude for 200 Hzfrequency for one hour followed by a 2 h rest interval. This 3 h patternis repeated for 48 hours with samples taken at 3, 6, 9, 24 and 48 h.These conditions are designed to mimic cyclic patterns during exercisetraining. This will help determine the role and rest intervals incardiac physiological adaptation, compared with the response seen inheart failure.

[0181] Once this mechanical stimulation limit is determined, theinventors will again measure rates of protein accumulation, cellgeometry and gene expression. The inventors will first examine theeffects of the stepped stretch protocol on the synthesis, turnover andgene expression of contractile proteins, as compared to theirnonstretched counterparts. Fractional synthetic rates (Ks, %/h) will bedetermined for MyHC and Kd values will be estimated in pulse-chaseexperiments. In addition, levels of mRNAs encoding genes up- ordown-regulated during cardiac hemodynamic overload in vivo will beexamined (Cadre et al., J. Mol. Cell. Cardiol. 30;2247-2259, 1998). Herethe inventors select members of the major contractile proteins (actin,α-MyHC, β-MyHC), the calcium release and uptake system (SERCA2, RyR),and a key focal adhesion signaling protein (FAK). Also, there may be adegree of stretch in the step protocol that induces gene expressionchanges characteristic of heart failure in vivo.

[0182] The original Flexercell (FX2000) had opaque membranes that didnot permit morphological studies. Nevertheless it had adhesivemembranes. The new Flex3000 (Bioflex) allows excellent morphologybecause the membranes are transparent but in introducing this superioroptical quality the adhesive properties were lost and the cells detachwhen mechanical deformed by the vacuum. Note that the Flexercell 3000device imposes a radial stretching of the membranes as it stretches thecircular membrane over the edge of the piston. The cells in the presentinvention are oriented in parallel arrays. This means that the cells onthe 12 to 6 O'clock axis are stretched longitudinally, whereas the cellson the 3 to 9 O'clock axis are stretched transversely. This would be abig problem for any study that scraped the cells and looked at averagedata. However, many of the inventors' morphological methods retaininformation on individual cells so that the two axes can be analyzedseparately from each membrane; one gives parallel strain and the othergives the perpendicular strain. The inventors contemplated production ofmembranes with concentric circles that would provide uniform transversestretch if the myocytes would follow the circular grooves. Suchmembranes may be used once they have been down sized to the microdomain. Membranes that have radial spokes also may be fabricated thatwould allow longitudinal strain to be experienced by all myocytes. Forsingle cell assay it is not an issue but for biochemical and molecularextraction methods where all cells are pooled, the inventors will useradial and concentric membranes.

[0183] The new microtextured surfaces will produce addition of newmyofibrils reproducing the hypertrophy observed in response to bothphysiological and pathological stimuli. These experiments will helpprovide a better understanding of the mechanisms involved inpathogenesis of heart failure and normal adaption to exercise.

[0184] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Incorporated herein by reference is Deutsch et al., J. Biomed.Mater. Res. (Appl. Biomater) 53:267-275, 2000, which provides additionalmethods that may be useful in conjunction with the present invention.

EXAMPLE 5 The Membranes of the Present Invention May be Used in ContactInhibition Investigations

[0185] Most normal cells and many cell lines do not grow indefinitely inthe body or in culture, rather they are inhibited by contact with theirneighbors; this state of arrest is known as contact inhibition. Forexample, melanoma cell lines can be cultured under conditions where theybecome inhibited by contact (Valyi-Nagy et al., 1993, Int. J. Cancer54:159-165), as can neural precursor lines transformed by polyoma largetumor (T) gene (Galiana et al., 1995, Proc. Natl. Acad. Sci. U.S.A.92:1560-1564), derivatives of colon HT29 cells (Velcich et al., 1995,Cell Growth Differ. 6:749-757), human umbilical vein endothelial cells(Gaits et al., Biochem. J. 311:97-103, 1995), nonparenchymal epithelialcells (Johnson et al., Cancer Lett. 96:37-48, 1995), and many others.

[0186] In the past, the phenomenon of contact inhibition of cells hasbeen used to select variants that continue to grow when saturation ofthe culture dish bottom has been reached. Foci have been isolated,comprised of cells that no longer respond to contact inhibitory signalsand are often more likely to form tumors in animals than their parentalcounterparts. Indeed, the initial identification of cellular oncogenesinvolved such an experimental approach. Land et al., 1983, Nature304:596-602; Copeland et al., 1979, Cell 17:993-1002.

[0187] In the present invention, the inventors have demonstrated thatthe membranes of the present invention can be used to model and studycontact inhibition. Seeing as loss of growth regulation of cells, e.g.,in cancer, is frequently reflected in the loss of contact inhibition ofcell proliferation, such models and studies will be useful inelucidating the mechanisms of such a loss of inhibition as well asproviding in vitro models which can be used to test various anti-canceragents.

[0188]FIG. 10A-D show phase images of cancer cell lines grown on 10 μMpegged silicone membranes and demonstrate that two kinds of cancer cellscan be grown on the membranes of the present invention but that cellresponse depends on the inherent properties of the type of cancer. Forexample, a cell line (Mum-2) that was derived from highly metastatic(invasive) cells in vivo differed from the cells derived from a solidtumor (Mel-1). FIG. 10A and 10B show non aggressive Mel-1 cells whichappear to attach to the pegs and cluster around the pegs. FIG. 10C and10D show the aggressive Mum-2 cells which appear to ignore the pegs andspread to distant locations on the membranes.

[0189] In order to demonstrate that cells grown on the membranes of thepresent invention undergo contact inhibition more than cells grown onconventional flat surfaces, the inventors seeded fibroblasts on peggedand flat silicon membranes and also on the microtextured membranes ofthe present invention. For determining the degree of cell attachment to10 μm pegs, fibroblast cells were cultured in Dulbecco's ModifiedEagle's medium with L-glutamine from the first passage of the primarymyocyte culture. Fibroblasts were plated at 20 cells per mm² and totalcell number was counted after trypsinisation over a 5 day period. InFIG. 11, fibroblast proliferation on 10 μM pegged (FIG. 11A) and flatsilicone membranes (FIG. 11B) is depicted at 5 days of growth. Thesefigures demonstrates the ability of the fibroblasts to exhibit reducedproliferation and growth (as indicated by increased cell number of cellsseeded on the pegged membranes as compared to the flat membrane) andalso demonstrates the tendency of the fibroblasts to extend filopediatowards and attachment to the pegs. The finding that the growth offibroblasts on microtextured silicone membrane is greatly reduced isshown in FIG. 12. Cell division was assessed by counting the number ofcells per dish and expressing this number as a percentage of the numberof cells on a flat silcone surface. FIG. 12 shows fibroblast cellproliferation per dish over a 5 day period. Note that at 5 days ofculture, fibroblast cell growth was 2 fold higher on flat membranescompared with pegged (p<0.005n=6 cultures).

[0190]FIG. 13 shows a Western blot of Cyclin D chosen as an indicator ofthe state of the cell cycle with respect to cell division. FIG. 13 showsthat cyclin D1 is 2.8 fold higher and significantly different (p<0.01)in fibroblast cultures grown on flat membranes after 48 hours of culturecompared with 10 micron pegged membranes. This shows that the pegmicro-topography alone blocks cell division by contact inhibition. Thisis in contrast to the conclusions reached by other researchers' studieswhich employed positive pegs and negative holes to observe fibroblastproliferation (Green et al., J Biomed Mater Res., 28(5)647-53, 1994).Green et al. found fluctuations between the flat, pegged and pittedsurfaces analyzed over a 12 day period, but these fluctuations seemed toindicate that the flat surface was consistently in the middle of thesurfaces analyzed. The data of Green et al. are most likely explained bythe fact that the projections and pits employed in that study are closerto each other than those of the present invention and as such the cells“see” those projections and pits as a flat surface.

[0191] The inventors found that fibroblast proliferation is decreased onthe microtextured surface(s) of the present invention has severalpotential implications. Current cell cultures are often overgrown withfibroblasts, a fact that may obscure potentially significantexperimental findings, by inundating cultures with fibroblast expressionlevels and not that of the particular cell of interest; the cardiaccontractile myocyte. By culturing the targeted cell on microtexturedsurfaces, this “contamination”, can be reduced significantly, therebyallowing any data acquired from such a culture to closer approximatewhat is occurring in, and representative of, the targeted contractilecell type.

[0192] Additionally, the microtextured surface of the present inventionwill be an invaluable tool in elucidating the signaling pathways and themechanisms responsible for the phenomenon of contact inhibition. As acomplement to this potential for studies on contact inhibition, theinventors envision the use of the surfaces of the present invention inefficient and quick screening procedures for cell biopsies of diseasedcell states that feature contact inhibition as one of theircharacteristics. Furthermore, the surfaces could prove invaluable indifferentiating among cell types within a more broadly defined group ofcells, such as cancer cells, by allowing rapid and efficientdetermination of which cancers are more likely to behave aggressively.This has already been demonstrated in the cancerous cell lines depictedin FIGS. 10A-10D.

[0193] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the invention. The entire disclosure of all publicationscited herein are hereby incorporated by reference.

1 6 1 6 PRT Fibronectin ligand receptor 1 Gly Arg Gly Asp Ser Pro 1 5 26 PRT Laminin ligand receptor 2 Tyr Ile Gly Ser Arg Cys 1 5 3 15 PRTSynthetic peptide 3 Cys Gly Gly Glu Gly Tyr Gly Glu Gly Arg Gly Asp SerPro Gly 1 5 10 15 4 4 PRT Synthetic peptide misc_feature (1)..(1) X =any amino acid 4 Xaa Arg Gly Asp 1 5 4 PRT Synthetic peptidemisc_feature (4) Xaa = any amino acid 5 Arg Gly Asp Xaa 1 6 5 PRTSynthetic peptide misc_feature (1) Xaa = any amino acid 6 Xaa Arg GlyAsp Xaa 1 5

We claim:
 1. A biocompatible, deformable membrane for the growth ofcells comprising a microtextured polymer membrane having projections ofbetween about 1 μm to about 100 μm in size and longitudinal grooves;wherein said polymer membrane comprises a surface modification tofacilitate cellular adhesion to said membrane, and further wherein saidgrowth of said cells on said membrane provides enhanced cellulardifferentiation of said cells as compared to growth on said polymermembrane in the absence of said grooves and/or said pegs.
 2. Themembrane of claim 1, wherein said polymer material is selected from thegroup consisting of silicone or other elastomeric polymers, hydrogels,biodegradables, bioerodible, or elastomeric polymers.
 3. The membrane ofclaim 1, wherein said surface modification comprises laminin,fibronectin, partial peptide sequences thereof or modifications oflaminin or fibronectin.
 4. The membrane of claim 1, wherein said cellsare muscle cells and the growth of said muscle cells on said membraneproduces muscle cells that have contractile function.
 5. A membrane ofclaim 1, wherein said membrane is fabricated into a master wafer using amethod selected from the group consisting of photolithography, diamondturning, diamond ruling and laser machining.
 6. A cell culture model forthe growth and development of cells comprising: the membrane of claim 1wherein said polymer membrane comprises a surface modification tofacilitate cellular adhesion to said membrane, wherein said membranecomprises surface microtopography to facilitate cellular orientation;and further wherein said growth of said cells on said membrane providesenhanced cellular differentiation of said cells as compared to growth onsaid polymer membrane in the absence of said grooves and pegs.
 7. Amethod of growing muscle cells comprising contacting a muscle cells withthe membrane of claim 1, under media conditions suitable to facilitatethe growth of said cell wherein growth of said muscle cells on saidmembrane reproduces the physiological micro-architecture of said musclecells.
 8. The method of claim 7 wherein said muscle cells are myocardialcells.
 9. The method of claim 7, wherein said muscle cells grown on saidmembrane are responsive to neurohormonal stimulation.
 10. The method ofclaim 7, wherein said muscle cells grown on said membrane exhibitcontractile function that mimic the contractile function of the musclecell in vivo, wherein said cells have mechanical deformation propertiesthat are similar to the mechanical deformation properties of said cellsin vivo.
 11. A method of producing a tissue patch comprising: (a)providing cells; (b) contacting said cells with the membrane of claim 1;(c) growing said cells in culture to allow the formation of tissue fromsaid cells.
 12. The method of claim 11, wherein said cell is selectedfrom the group consisting of skeletal muscle, smooth muscle, cardiacmuscle, vascular endothelial cells, lymphatic endothelial cells, stemcells, endothelial cartilage, bone cells or other cell types stimulatedby mechanical force or subject to contact inhibition.